Microorganisms for producing methacrylic acid and methacrylate esters and methods related thereto

ABSTRACT

The invention provides a non-naturally occurring microbial organism having a methacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate pathway. The microbial organism contains at least one exogenous nucleic acid encoding an enzyme in a methacrylic acid pathway. The invention additionally provides a method for producing methacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate. The method can include culturing methacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate producing microbial organism, where the microbial organism expresses at least one exogenous nucleic acid encoding a methacrylic acid pathway enzyme in a sufficient amount to produce methacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate, under conditions and for a sufficient period of time to produce methacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate.

This application claims the benefit of priority of U.S. Provisionalapplication Ser. No. 61/471,078, filed Apr. 1, 2011, and U.S.Provisional application Ser. No. 61/571,232, filed Jun. 22, 2011, andU.S. Provisional application Ser. No. 61/509,560, filed Jul. 19, 2011,and U.S. Provisional application Ser. No. 61/510,054, filed Jul. 20,2011, and U.S. Provisional application Ser. No. 61/512,348, filed Jul.27, 2011, each of which the entire contents are incorporated herein byreference.

Incorporated herein by reference is the Sequence Listing being submittedvia EFS-Web as an ASCII text file named12956-122-999_SequenceListing.TXT, created Nov. 9, 2012, and being78,078 bytes in size.

BACKGROUND OF THE INVENTION

The present invention relates generally to biosynthetic processes, andmore specifically to organisms having methacrylic acid biosyntheticcapabilities.

Methyl methacrylate (MMA) is an organic compound with the formulaCH₂═C(CH₃)CO₂CH₃. This colourless liquid is the methyl ester ofmethacrylic acid (MMA) and is the monomer for the production of thetransparent plastic polymethyl methacrylate (PMMA).

The principal application of methyl methacrylate is the production ofpolymethyl methacrylate acrylic plastics. Also, methyl methacrylate isused for the production of the co-polymer methylmethacrylate-butadiene-styrene (MBS), used as a modifier for PVC. Methylmethacrylate polymers and co-polymers are used for waterborne coatings,such as latex paint. Uses are also found in adhesive formulations.Contemporary applications include the use in plates that keep lightspread evenly across liquid crystal display (LCD) computer and TVscreens. Methyl methacrylate is also used to prepare corrosion casts ofanatomical organs, such as coronary arteries of the heart.

Methacrylic acid, or 2-methyl-2-propenoic acid, is a low molecularweight carboxylic acid that occurs naturally in small amounts in the oilof Roman chamomile. It is a corrosive liquid with an acrid unpleasantodor. It is soluble in warm water and miscible with most organicsolvents. Methacrylic acid polymerizes readily upon heating or treatmentwith a catalytic amount of strong acid, such as HCl. The resultingpolymer is a ceramic-looking plastic. Methacrylic acid is usedindustrially in the preparation of its esters, known collectively asmethacrylates, such as methyl methacrylate. The methacrylates havenumerous uses, most notably in the manufacture of polymers.

Most commercial producers apply an acetone cyanohydrin (ACH) route toproduce methacrylic acid (MAA), with acetone and hydrogen cyanide as rawmaterials. The intermediate cyanohydrin is converted with sulfuric acidto a sulfate ester of the methacrylamide, hydrolysis of which givesammonium bisulfate and MAA. Some producers start with an isobutylene or,equivalently, tert-butanol, which is oxidized to methacrolein, and againoxidized to methacrylic acid. MAA is then esterified with methanol toMMA.

The conventional production process, using the acetone cyanohydrinroute, involves the conversion of hydrogen cyanide (HCN) and acetone toacetone cyanohydrin, which then undergoes acid assisted hydrolysis andesterification with methanol to give MMA. Difficulties in handlingpotentially deadly HCN along with the high costs of byproduct disposal(1.2 tons of ammonium bisulfate are formed per ton of MMA) have sparkeda great deal of research aimed at cleaner and more economical processes.A number of new processes have been commercialized over the last twodecades and many more are close to commercialization. The Asahi “DirectMetha” route, which involves the oxidation of isobutylene tomethacrolein, which is then mixed with methanol, oxidized with air, andesterified to MMA, has been described as an economical process.

Other than MMA polymers, the other major product of this industry iscrude methacrylic acid, which accounts for about 20 percent of the totalproduction of MMA. Crude MAA is processed into butyl methacrylatesand/or “glacial” MAA, which is highly purified crude MAA. Glacial MAAcan be used directly as a comonomer in various polymers and is also usedto make a variety of small volume methacrylates. On the other hand, MAAcan also be converted into MMA via esterification with methanol.

Thus, there exists a need for alternative methods for effectivelyproducing compounds such as methacrylic acid. The present inventionsatisfies this need and provides related advantages as well.

SUMMARY OF INVENTION

The invention provides a non-naturally occurring microbial organismhaving a methacrylic acid, methacrylate ester such as methylmethacrylate, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate pathway.The microbial organism contains at least one exogenous nucleic acidencoding an enzyme in a methacrylic acid pathway. The inventionadditionally provides a method for producing methacrylic acid,methacrylate ester such as methyl methacrylate, 3-hydroxyisobutyrateand/or 2-hydroxyisobutyrate. The method can include culturingmethacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate producing microbial organism, where the microbialorganism expresses at least one exogenous nucleic acid encoding amethacrylic acid pathway enzyme in a sufficient amount to producemethacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate, under conditions and for a sufficient period oftime to produce methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate.

The invention also provides non-naturally occurring microbial organismscontaining methacrylate ester or methyl methacrylate pathways comprisingat least one exogenous nucleic acid encoding a methacrylate ester or amethyl methacrylate enzyme expressed in a sufficient amount to producemethacrylate ester or methyl methacrylate. The microbial organismcontains at least one exogenous nucleic acid encoding an enzyme in themethacrylate ester or methyl methacrylate pathway and at least oneexogenous nucleic acid that encodes an enzyme that increases the yieldsof methacrylate ester or methyl methacrylate by (i) enhancing carbonfixation via the reductive TCA cycle, and/or (ii) accessing additionalreducing equivalents from gaseous carbon sources and/or syngascomponents such as CO, CO₂, and/or H₂. In some aspects, thenon-naturally occurring microbial organisms comprise (i) a reductive TCApathway comprising at least one exogenous nucleic acid encoding areductive TCA pathway enzyme, wherein the at least one exogenous nucleicacid is selected from an ATP-citrate lyase, a citrate lyase, a fumaratereductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) areductive TCA pathway comprising at least one exogenous nucleic acidencoding a reductive TCA pathway enzyme, wherein the at least oneexogenous nucleic acid is selected from a pyruvate:ferredoxinoxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvatecarboxykinase, a CO dehydrogenase, and an H₂ hydrogenase; or (iii) atleast one exogenous nucleic acid encodes an enzyme selected from a COdehydrogenase, an H₂ hydrogenase, and combinations thereof. Theinvention additionally provides methods of using such microbialorganisms to produce methacrylate ester or methyl methacrylate, byculturing a non-naturally occurring microbial organism containingmethacrylate ester or methyl methacrylate pathways under conditions andfor a sufficient period of time to produce methacrylate ester or methylmethacrylate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows exemplary pathways to methacrylic acid from pyruvate andacetyl-CoA, and aconitate. The enzymes are A. citramalate synthase, B.citramalate dehydratase (citraconate forming), C. citraconatedecarboxylase, D. citramalyl-CoA lyase, E. citramalyl-CoA transferase,synthetase or hydrolase, F. citramalate dehydratase (mesaconateforming), G. citraconate isomerase, H. mesaconate decarboxylase, I.aconitate decarboxylase, J. itaconate isomerase, K. citramalyl-CoAdehydratase and L. itaconyl-CoA transferase, synthetase or hydrolase.

FIG. 2 shows exemplary pathways from methacrylate to methacrylateesters. Methacrylate can be formed from the pathways depicted in FIG. 1or other methacrylate pathways such as those described in WO 2009/135074and U.S. publication 2009/0275096. Methacrylyl-CoA can be formed frommethacrylate via a methacrylyl-CoA transferase or a methacrylyl-CoAsynthetase. Methacrylate esters can be formed from methacrylyl-CoA andan alcohol in the presence of an alcohol transferase enzyme.Methacrylate esters can also be formed directly from methacrylate and analcohol by a methacrylate ester-forming enzyme or by chemical conversion(for example, heating in the presence of a dehydrating agent such as anacid). R denotes any organic functional group including, but not limitedto, a methyl, ethyl, n-propyl, n-butyl, i-propyl, sec-butyl, andtert-butyl, pentyl, or hexyl functional group. For example, if R denotesa methyl-group, R—OH denotes methanol and the product of the pathway ismethylmethacrylate.

FIG. 3 shows an exemplary metabolic pathway from succinyl-CoA to MMA via3-hydroxyisobutyrate.

FIG. 4 shows an exemplary succinyl-CoA to MAA pathway via3-amino-2-methylpropionate. The “lumped reaction” (steps 2-3) iscatalyzed by 1) methylmalonyl-CoA epimerase and 2) methylmalonyl-CoAreductase.

FIG. 5 shows an exemplary 4-hydroxybutyryl-CoA to MAA pathway thatproceeds via 3-hydroxyisobutyrate or methacrylyl-CoA. Step 2 can becatalyzed by three alternative enzymes: 3-hydroxyisobutyryl-CoAsynthetase, 3-hydroxyisobutyryl-CoA hydrolase or 3-hydroxyisobutyryl-CoAtransferase. Similarly, step 5 can be catalyzed by three alternativeenzymes:methacrylyl-CoA synthetase, methacrylyl-CoA hydrolase ormethacrylyl-CoA transferase.

FIG. 6 shows an exemplary alpha-ketoglutarate to MAA pathway viathreo-3-methylaspartate.

FIG. 7 shows an exemplary alpha-ketoglutarate to MAA pathway via2-hydroxyglutarate.

FIG. 8 shows exemplary metabolic pathways for the conversion ofacetyl-CoA or 4-hydroxybutyryl-CoA into MAA or 2-hydroxyisobutyrate.

FIG. 9 shows an exemplary pathway from acetyl-CoA to MAA.

FIG. 10 shows an exemplary pyruvate to acrylyl-CoA to MAA pathway.

FIG. 11 shows an exemplary 2-ketovalerate to MAA pathway.2-Ketoisovalerate can be produced either from valine or pyruvate. Anexemplary set of enzymes for pyruvate conversion to 2-ketoisovalerate iscomprised of acetolactate synthase, acetohydroxy acid isomeroreductase,and dihydroxyacid dehydratase.

FIG. 12 shows the reverse TCA cycle for fixation of CO₂ on carbohydratesas substrates. The enzymatic transformations are carried out by theenzymes as shown.

FIG. 13 shows the pathway for the reverse TCA cycle coupled with carbonmonoxide dehydrogenase and hydrogenase for the conversion of syngas toacetyl-CoA.

FIG. 14 shows Western blots of 10 micrograms ACS90 (lane 1), ACS91(lane2), Mta98/99 (lanes 3 and 4) cell extracts with size standards(lane 5) and controls of M. thermoacetica CODH (Moth_(—)1202/1203) orMtr (Moth_(—)1197) proteins (50, 150, 250, 350, 450, 500, 750, 900, and1000 ng).

FIG. 15 shows CO oxidation assay results. Cells (M. thermoacetica or E.coli with the CODH/ACS operon; ACS90 or ACS91 or empty vector: pZA33S)were grown and extracts prepared. Assays were performed at 55° C. atvarious times on the day the extracts were prepared. Reduction ofmethylviologen was followed at 578 nm over a 120 sec time course.

FIG. 16A shows the pathways for the biosynthesis of methacrylic acid and3-hydroxyisobutyric acid. The enzymatic transformations shown arecarried out by the following enzymes: (1) hydrogenase, (2) carbonmonoxide dehydrogenase, (3) succinyl-CoA transferase, succinyl-CoAsynthetase, (4) methylmalonyl-CoA mutase, (5) methylmalonyl-CoAepimerase, (6) methylmalonyl-CoA reductase, (7) methylmalonatesemialdehyde reductase and (8) 3-hydroxyisobutyrate dehydratase.

FIG. 16B shows the pathway for biosynthesis of methacrylic acid fromglucose via a 4-hydroxybutyryl-CoA intermediate. The enzymatictransformations are carried out by the enzymes: (1) hydrogenase, (2)carbon monoxide dehydrogenase, (3) succinyl-CoA transferase,succinyl-CoA synthetase, (4) succinyl-CoA reductase (aldehyde forming),(5) 4-hydroxybutyrate dehydrogenase, (6) 4-hydroxybutyrate kinase, (7)phosphotrans-4-hydroxybutyrylase, (8) succinate reductase, (9)succinyl-CoA reductase (alcohol forming), (10) 4-hydroxybutyryl-CoAsynthetase, 4-hydroxybutyryl-CoA transferase, (11) 4-hydroxybutyryl-CoAmutase, (12) 3-hydroxyisobutyryl-CoA synthetase, transferase orhydrolase, (13) 3-hydroxyisobutyrate dehydratase, (14)3-hydroxyisobutyryl-CoA dehydratase, (15) methacrylyl-CoA synthetase,transferase or hydrolase.

FIG. 16C shows the flux distribution showing an enhanced maximumtheoretical yield of methacrylic acid on glucose when carbon is routedvia the reductive TCA cycle. The enzymatic transformations are carriedout by: (1) ATP-citrate lyase; citrate lyase, acetyl-CoA synthetase; orcitrate lyase, acetate kinse, phosphotransacetylase, (2) malatedehydrogenase, (3) fumarase, (4) fumarate reductase, (5) succinyl-CoAsynthetase or transferase, (6) alpha-ketoglutarate:ferridoxinoxidoreductase, (7) isocitrate dehydrogenase, (8) aconitase, (9)pyruvate:ferridoxin oxidoreductase; pyruvate oxidase, acetyl-CoAsynthetase; or pyruvate oxidase, acetate kinase, phosphotransacetylase,(10) acetoacetyl-CoA thiolase, (11) acetoacetyl-CoA reductase, (12)3-hydroxybutyryl-CoA mutase, (13) 2-hydroxybutyryl-CoA dehydratase, (14)methacrylyl-CoA synthetase, transferase or hydrolase, (15)2-hydroxyisobutyryl-CoA synthetase, transferase or hydrolase.

FIG. 16D shows the flux distribution showing an enhanced maximumtheoretical yield of methacrylic acid on glucose via citramalate whencarbon is routed via the reductive TCA cycle. The enzymatictransformations are carried out by (1) ATP-citrate lyase; citrate lyase,acetyl-CoA synthetase; or citrate lyase, acetate kinse,phosphotransacetylase, (2) malate dehydrogenase, (3) fumarase, (4)fumarate reductase, (5) succinyl-CoA synthetase or transferase, (6)alpha-ketoglutarate:ferridoxin oxidoreductase, (7) isocitratedehydrogenase, (8) aconitase, (9) pyruvate:ferridoxin oxidoreductase;pyruvate oxidase, acetate kinase, phosphotransacetylase, (10)citramalate synthase, (11) citramalate dehydratase and (12) citraconatedecarboxylase.

FIG. 16E shows pathways for biosynthesis of methacrylic acid fromacetyl-CoA and pyruvate. The enzymatic transformations are carried outby the enzymes as shown. Enzymes are (1) citramalate synthase, (2)citramalate dehydratase (citraconate forming), (3) citraconatedecarboxylase, (4) citramalyl-CoA lyase, (5) citramalyl-CoA transferase,synthetase or hydrolase, (6) citramalate dehydratase (mesaconateforming), (7) citraconate isomerase, (8) mesaconate decarboxylase, (9)aconitate decarboxylase, (10) itaconate isomerase, (11) itaconyl-CoAtransferase or synthetase and (12) itaconyl-CoA hydratase.

FIGS. 17A and 17B show exemplary pathways. The enzymatic transformationsare carried out by the enzymes as shown. FIG. 17A shows the pathways forfixation of CO₂ to succinyl-CoA using the reductive TCA cycle. FIG. 17Bshows exemplary pathways for the biosynthesis of 3-hydroxyisobutric acidand methacrylic acid from succinyl-CoA; the enzymatic transformationsshown are carried out by the following enzymes: A. Methylmalonyl-CoAmutase, B. Methylmalonyl-CoA epimerase, C. Methylmalonyl-CoA reductase,D. Methylmalonate semialdehyde reductase, E. 3-Hydroxyisobutyratedehydratase.

FIGS. 18A and 18B show exemplary pathways. The enzymatic transformationsare carried out by the enzymes as shown. FIG. 18A shows the pathways forfixation of CO₂ to succinate using the reductive TCA cycle. FIG. 18Bshows exemplary pathways for the biosynthesis of 3-hydroxyisobutyricacid and methacrylic acid from succinate; the enzymatic transformationsshown are carried out by the following enzymes: A.3-Hydroxyisobutyryl-CoA dehydratase, B. Methacrylyl-CoA synthetase,transferase or hydrolase, C. Succinyl-CoA transferase or synthetase, D.Succinyl-CoA reductase (aldehyde forming), E. 4-Hydroxybutyratedehydrogenase, F. 4-Hydroxybutyrate kinase, G.Phosphotrans-4-hydroxybutyrylase, H. Succinate reductase, I.Succinyl-CoA reductase (alcohol forming), J. 4-Hydroxybutyryl-CoAsynthetase or transferase, K. 4-Hydroxybutyryl-CoA mutase, L.3-Hydroxyisobutyryl-CoA synthetase, transferase or hydrolase, M.3-Hydroxyisobutyrate dehydratase.

FIGS. 19A and 19B show exemplary pathways. FIG. 19A shows the pathwaysfor fixation of CO₂ to acetyl-CoA and pyruvate using the reductive TCAcycle. FIG. 19B shows exemplary pathways for the biosynthesis ofmethacrylate from acetyl-CoA and pyruvate; the enzymatic transformationsshown are carried out by the following enzymes: 1. Citramalate synthase,2. Citramalate dehydratase (citraconate forming), 3. Citraconatedecarboxylase, 4. Citramalyl-CoA lyase, 5. Citramalyl-CoA transferase,synthetase or hydrolase, 6. Citramalate dehydratase (mesaconateforming), 7. Citraconate isomerase, 8. Mesaconate decarboxylase, 9.Aconitate decarboxylase, 10. Itaconate isomerase, 11. Itaconyl-CoAtransferase or synthetase, 12. Itaconyl-CoA hydratase.

FIGS. 20A and 20B show exemplary pathways. FIG. 20A shows the pathwaysfor fixation of CO₂ to acetyl-CoA using the reductive TCA cycle. FIG.20B shows exemplary pathways for the biosynthesis of methacrylic acidand 2-hydroxyisobutyric acid from acetyl-CoA.

FIGS. 21A and 21B show exemplary pathways. FIG. 21A shows the pathwaysfor fixation of CO2 to acetyl-CoA using the reductive TCA cycle. FIG.21B shows exemplary pathways for the biosynthesis of methacrylic acidand 3-hydroxyisobutyric acid from acetyl-CoA; the enzymatictransformations shown are carried out by the following enzymes: 1)Acetoacetyl-CoA thiolase (AtoB), 2) 3-Hydroxybutyryl-CoA dehydrogenase(Hbd), 3) Crotonase (Crt), 4) Crotonyl-CoA hydratase (4-Budh), 5)4-hydroxybutyryl-CoA mutase, 6) 3-hydroxyisobutyryl-CoA hydrolase,synthetase, or transferase, 7) 3-hydroxyisobutyric acid dehydratase, 8)3-hydroxyisobutyryl-CoA dehydratase, 9) methacrylyl-CoA hydrolase,synthetase, or transferase.

FIG. 22A shows the nucleotide sequence (SEQ ID NO:1) of carboxylic acidreductase from Nocardia iowensis (GNM 720), and FIG. 22B shows theencoded amino acid sequence (SEQ ID NO:2).

FIG. 23A shows the nucleotide sequence (SEQ ID NO:3) ofphosphpantetheine transferase, which was codon optimized, and FIG. 23Bshows the encoded amino acid sequence (SEQ ID NO:4).

FIG. 24A shows the nucleotide sequence (SEQ ID NO:5) of carboxylic acidreductase from Mycobacterium smegmatis mc(2)155 (designated 890), andFIG. 24B shows the encoded amino acid sequence (SEQ ID NO:6).

FIG. 25A shows the nucleotide sequence (SEQ ID NO:7) of carboxylic acidreductase from Mycobacterium avium subspecies paratuberculosis K-10(designated 891), and FIG. 25B shows the encoded amino acid sequence(SEQ ID NO:8).

FIG. 26A shows the nucleotide sequence (SEQ ID NO:9) of carboxylic acidreductase from Mycobacterium marinum M (designated 892), and FIG. 26Bshows the encoded amino acid sequence (SEQ ID NO:10).

FIG. 27A shows the nucleotide sequence (SEQ ID NO:11) of carboxylic acidreductase designated 891GA, and FIG. 27B shows the encoded amino acidsequence (SEQ ID NO:12).

FIG. 28 shows an exemplary pathway to a methacrylate ester via3-hydroxyisobutyrate and/or 3-hydroxyisobutyryl-CoA. R—OH refers to anyorganic alcohol.

FIG. 29 shows an exemplary pathway to a methacrylate ester via2-hydroxyisobutyrate and/or 2-hydroxyisobutyryl-CoA. R—OH refers to anyorganic alcohol.

FIG. 30 shows an exemplary pathway to an exemplary methacrylate ester,methyl methacrylate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the design and production of cellsand organisms having biosynthetic production capabilities formethacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate. As disclosed herein, metabolic pathways can bedesigned and recombinantly engineered to achieve the biosynthesis ofmethacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate in microbial organisms such as Escherichia coli andother cells or organisms. Biosynthetic production of methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate isconfirmed by construction of strains having the designed metabolicgenotype. These metabolically engineered cells or organisms also can besubjected to adaptive evolution to further augment methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyratebiosynthesis, including under conditions approaching theoretical maximumgrowth.

Microorganisms for the production of methacrylic acid (MAA) have beenpreviously described (see, for example, WO 2009/135074 and U.S.publication 2009/0275096, which is incorporated herein by reference).The present invention provides additional pathways for engineeringmicrobial organisms for the production of methacrylic acid (MAA). FIG. 1provides exemplary pathways to MAA from acetyl-CoA and pyruvate viaintermediate citramalate. Also shown are pathways to MAA from aconitate.In one pathway, acetyl-CoA and pyruvate are first converted tocitramalate by citramalate synthase. Dehydration of citramalate canyield either citraconate (Step B) or mesaconate (Step C). Mesaconate andcitraconate are interconverted by a cis/trans isomerase in Step G.Decarboxylation of mesaconate (Step H) or citraconate (Step C) yieldsMAA. In an alternate pathway, citramalate is formed from acetyl-CoA andpyruvate via a citramalyl-CoA intermediate in Steps D and E, catalyzedby citramalyl-CoA lyase and citramalyl-coA hydrolase, transferase orsynthetase.

The invention also encompasses pathways from aconitate to MAA. In onepathway, aconitate is first decarboxylated to itaconate by aconitatedecarboxylase (Step I). Itaconate is then isomerized to citraconate byitaconate delta-isomerase (Step J). Conversion of citraconate to MAAproceeds either directly by decarboxylation or indirectly viamesaconate. In an alternate pathway, the itaconate intermediate is firstconverted to itaconyl-CoA by a CoA transferase or synthetase (Step L).Hydration of itaconyl-CoA yields citramalyl-CoA, which can then beconverted to MAA as described previously. Additionally details andembodiments of the exemplary MAA pathways are described herein below.

As used herein, the term “non-naturally occurring” when used inreference to a microbial organism or microorganism of the invention isintended to mean that the microbial organism has at least one geneticalteration not normally found in a naturally occurring strain of thereferenced species, including wild-type strains of the referencedspecies. Genetic alterations include, for example, modificationsintroducing expressible nucleic acids encoding metabolic polypeptides,other nucleic acid additions, nucleic acid deletions and/or otherfunctional disruption of the microbial organism's genetic material. Suchmodifications include, for example, coding regions and functionalfragments thereof, for heterologous, homologous or both heterologous andhomologous polypeptides for the referenced species. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a gene or operon. Exemplarymetabolic polypeptides include enzymes or proteins within a methacrylicacid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate biosynthetic pathway.

A metabolic modification refers to a biochemical reaction that isaltered from its naturally occurring state. Therefore, non-naturallyoccurring microorganisms can have genetic modifications to nucleic acidsencoding metabolic polypeptides, or functional fragments thereof.Exemplary metabolic modifications are disclosed herein.

As used herein, the term “isolated” when used in reference to amicrobial organism is intended to mean an organism that is substantiallyfree of at least one component as the referenced microbial organism isfound in nature. The term includes a microbial organism that is removedfrom some or all components as it is found in its natural environment.The term also includes a microbial organism that is removed from some orall components as the microbial organism is found in non-naturallyoccurring environments. Therefore, an isolated microbial organism ispartly or completely separated from other substances as it is found innature or as it is grown, stored or subsisted in non-naturally occurringenvironments. Specific examples of isolated microbial organisms includepartially pure microbes, substantially pure microbes and microbescultured in a medium that is non-naturally occurring.

As used herein, the terms “microbial,” “microbial organism” or“microorganism” are intended to mean any organism that exists as amicroscopic cell that is included within the domains of archaea,bacteria or eukarya. Therefore, the term is intended to encompassprokaryotic or eukaryotic cells or organisms having a microscopic sizeand includes bacteria, archaea and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. The term alsoincludes cell cultures of any species that can be cultured for theproduction of a biochemical.

As used herein, “methacrylic acid,” having the chemical formulaCH₂═C(CH₃)CO₂ (see FIG. 1) (IUPAC name 2-methyl-2-propenoic acid), isthe acid form of methacrylate, and it is understood that methacrylicacid and methacrylate can be used interchangeably throughout to refer tothe compound in any of its neutral or ionized forms, including any saltforms thereof. It is understood by those skilled understand that thespecific form will depend on the pH.

As used herein, “methacrylate ester,” refers to a compound having thechemical formula CH₂═C(CH₃)COOR (see FIGS. 28 and 29), wherein R is alower alkyl, that is C1 to C6, branched or straight chain, including,without limitation, methyl, ethyl, n-propyl, n-butyl, i-propyl,sec-butyl, and tert-butyl, pentyl, or hexyl, any of which can beunsaturation thereby being, for example, propenyl, butenyl, pentyl, andhexenyl. Exemplary methacrylate esters include, without limitation,methyl methacrylate, ethyl methacrylate, and n-propyl methacrylate.Methacrylate esters as used herein also include other R groups that aremedium to long chain groups, that is C7-C22, wherein the methacrylateesters are derived from fatty alcohols, such as heptyl, octyl, nonyl,decyl, undecyl, lauryl, tridecyl, myristyl, pentadecyl, cetyl,palmitolyl, heptadecyl, stearyl, nonadecyl, arachidyl, heneicosyl, andbehenyl alcohols, any one of which can be optionally branched and/orcontain unsaturations.

As used herein, the term “CoA” or “coenzyme A” is intended to mean anorganic cofactor or prosthetic group (nonprotein portion of an enzyme)whose presence is required for the activity of many enzymes (theapoenzyme) to form an active enzyme system. Coenzyme A functions incertain condensing enzymes, acts in acetyl or other acyl group transferand in fatty acid synthesis and oxidation, pyruvate oxidation and inother acetylation.

As used herein, the term “substantially anaerobic” when used inreference to a culture or growth condition is intended to mean that theamount of oxygen is less than about 10% of saturation for dissolvedoxygen in liquid media. The term also is intended to include sealedchambers of liquid or solid medium maintained with an atmosphere of lessthan about 1% oxygen.

“Exogenous” as it is used herein is intended to mean that the referencedmolecule or the referenced activity is introduced into the hostmicrobial organism. The molecule can be introduced, for example, byintroduction of an encoding nucleic acid into the host genetic materialsuch as by integration into a host chromosome or as non-chromosomalgenetic material such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe microbial organism. When used in reference to a biosyntheticactivity, the term refers to an activity that is introduced into thehost reference organism. The source can be, for example, a homologous orheterologous encoding nucleic acid that expresses the referencedactivity following introduction into the host microbial organism.Therefore, the term “endogenous” refers to a referenced molecule oractivity that is present in the host. Similarly, the term when used inreference to expression of an encoding nucleic acid refers to expressionof an encoding nucleic acid contained within the microbial organism. Theterm “heterologous” refers to a molecule or activity derived from asource other than the referenced species whereas “homologous” refers toa molecule or activity derived from the host microbial organism.Accordingly, exogenous expression of an encoding nucleic acid of theinvention can utilize either or both a heterologous or homologousencoding nucleic acid.

It is understood that when more than one exogenous nucleic acid isincluded in a microbial organism that the more than one exogenousnucleic acids refers to the referenced encoding nucleic acid orbiosynthetic activity, as discussed above. It is further understood, asdisclosed herein, that such more than one exogenous nucleic acids can beintroduced into the host microbial organism on separate nucleic acidmolecules, on polycistronic nucleic acid molecules, or a combinationthereof, and still be considered as more than one exogenous nucleicacid. For example, as disclosed herein a microbial organism can beengineered to express two or more exogenous nucleic acids encoding adesired pathway enzyme or protein. In the case where two exogenousnucleic acids encoding a desired activity are introduced into a hostmicrobial organism, it is understood that the two exogenous nucleicacids can be introduced as a single nucleic acid, for example, on asingle plasmid, on separate plasmids, can be integrated into the hostchromosome at a single site or multiple sites, and still be consideredas two exogenous nucleic acids. Similarly, it is understood that morethan two exogenous nucleic acids can be introduced into a host organismin any desired combination, for example, on a single plasmid, onseparate plasmids, can be integrated into the host chromosome at asingle site or multiple sites, and still be considered as two or moreexogenous nucleic acids, for example three exogenous nucleic acids.Thus, the number of referenced exogenous nucleic acids or biosyntheticactivities refers to the number of encoding nucleic acids or the numberof biosynthetic activities, not the number of separate nucleic acidsintroduced into the host organism.

The non-naturally occurring microbial organisms of the invention cancontain stable genetic alterations, which refers to microorganisms thatcan be cultured for greater than five generations without loss of thealteration. Generally, stable genetic alterations include modificationsthat persist greater than 10 generations, particularly stablemodifications will persist more than about 25 generations, and moreparticularly, stable genetic modifications will be greater than 50generations, including indefinitely.

Those skilled in the art will understand that the genetic alterations,including metabolic modifications exemplified herein, are described withreference to a suitable host organism such as E. coli and theircorresponding metabolic reactions or a suitable source organism fordesired genetic material such as genes for a desired metabolic pathway.However, given the complete genome sequencing of a wide variety oforganisms and the high level of skill in the area of genomics, thoseskilled in the art will readily be able to apply the teachings andguidance provided herein to essentially all other organisms. Forexample, the E. coli metabolic alterations exemplified herein canreadily be applied to other species by incorporating the same oranalogous encoding nucleic acid from species other than the referencedspecies. Such genetic alterations include, for example, geneticalterations of species homologs, in general, and in particular,orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent andare responsible for substantially the same or identical functions indifferent organisms. For example, mouse epoxide hydrolase and humanepoxide hydrolase can be considered orthologs for the biologicalfunction of hydrolysis of epoxides. Genes are related by verticaldescent when, for example, they share sequence similarity of sufficientamount to indicate they are homologous, or related by evolution from acommon ancestor. Genes can also be considered orthologs if they sharethree-dimensional structure but not necessarily sequence similarity, ofa sufficient amount to indicate that they have evolved from a commonancestor to the extent that the primary sequence similarity is notidentifiable. Genes that are orthologous can encode proteins withsequence similarity of about 25% to 100% amino acid sequence identity.Genes encoding proteins sharing an amino acid similarity less that 25%can also be considered to have arisen by vertical descent if theirthree-dimensional structure also shows similarities. Members of theserine protease family of enzymes, including tissue plasminogenactivator and elastase, are considered to have arisen by verticaldescent from a common ancestor.

Orthologs include genes or their encoded gene products that through, forexample, evolution, have diverged in structure or overall activity. Forexample, where one species encodes a gene product exhibiting twofunctions and where such functions have been separated into distinctgenes in a second species, the three genes and their correspondingproducts are considered to be orthologs. For the production of abiochemical product, those skilled in the art will understand that theorthologous gene harboring the metabolic activity to be introduced ordisrupted is to be chosen for construction of the non-naturallyoccurring microorganism. An example of orthologs exhibiting separableactivities is where distinct activities have been separated intodistinct gene products between two or more species or within a singlespecies. A specific example is the separation of elastase proteolysisand plasminogen proteolysis, two types of serine protease activity, intodistinct molecules as plasminogen activator and elastase. A secondexample is the separation of mycoplasma 5′-3′ exonuclease and DrosophilaDNA polymerase III activity. The DNA polymerase from the first speciescan be considered an ortholog to either or both of the exonuclease orthe polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by, for example, duplicationfollowed by evolutionary divergence and have similar or common, but notidentical functions. Paralogs can originate or derive from, for example,the same species or from a different species. For example, microsomalepoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase(epoxide hydrolase II) can be considered paralogs because they representtwo distinct enzymes, co-evolved from a common ancestor, that catalyzedistinct reactions and have distinct functions in the same species.Paralogs are proteins from the same species with significant sequencesimilarity to each other suggesting that they are homologous, or relatedthrough co-evolution from a common ancestor. Groups of paralogousprotein families include HipA homologs, luciferase genes, peptidases,and others.

A nonorthologous gene displacement is a nonorthologous gene from onespecies that can substitute for a referenced gene function in adifferent species. Substitution includes, for example, being able toperform substantially the same or a similar function in the species oforigin compared to the referenced function in the different species.Although generally, a nonorthologous gene displacement will beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless will still fallwithin the meaning of the term as it is used herein. Functionalsimilarity requires, for example, at least some structural similarity inthe active site or binding region of a nonorthologous gene productcompared to a gene encoding the function sought to be substituted.Therefore, a nonorthologous gene includes, for example, a paralog or anunrelated gene.

Therefore, in identifying and constructing the non-naturally occurringmicrobial organisms of the invention having methacrylic acidbiosynthetic capability, those skilled in the art will understand withapplying the teaching and guidance provided herein to a particularspecies that the identification of metabolic modifications can includeidentification and inclusion or inactivation of orthologs. To the extentthat paralogs and/or nonorthologous gene displacements are present inthe referenced microorganism that encode an enzyme catalyzing a similaror substantially similar metabolic reaction, those skilled in the artalso can utilize these evolutionally related genes.

Orthologs, paralogs and nonorthologous gene displacements can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides will reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences.

Exemplary parameters for determining relatedness of two or moresequences using the BLAST algorithm, for example, can be as set forthbelow. Briefly, amino acid sequence alignments can be performed usingBLASTP version 2.0.8 (Jan. 5.1999) and the following parameters: Matrix:0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50; expect: 10.0;wordsize: 3; filter: on. Nucleic acid sequence alignments can beperformed using BLASTN version 2.0.6 (Sep. 16.1998) and the followingparameters: Match: 1; mismatch: −2; gap open: 5; gap extension: 2;x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off. Those skilled inthe art will know what modifications can be made to the above parametersto either increase or decrease the stringency of the comparison, forexample, and determine the relatedness of two or more sequences.

In one embodiment, the invention provides a non-naturally occurringmicrobial organism having a methacrylate ester pathway comprising atleast one exogenous nucleic acid encoding a methacrylate ester pathwayenzyme expressed in a sufficient amount to produce a methacrylate ester,where the methacrylate ester pathway comprises an alcohol transferase oran ester-forming enzyme, and a dehydratase. In a particular embodiment,the microbial organism comprises two exogenous nucleic acids eachencoding a methacrylate ester pathway enzyme. For example, the twoexogenous nucleic acids can encode an alcohol transferase and adehydratase or alternatively an ester-forming enzyme and a dehydratase.In a particular embodiment, the at least one exogenous nucleic acid canbe a heterologous nucleic acid. In another embodiment, the non-naturallyoccurring microbial organism can be in a substantially anaerobic culturemedium. The invention additionally provides a method for producingmethacrylate ester, comprising culturing the non-naturally occurringmicrobial organism disclosed herein having a methacrylate ester pathwayunder conditions and for a sufficient period of time to producemethacrylate ester.

The invention additionally provides a non-naturally occurring microbialorganism having a methyl methacrylate pathway comprising at least oneexogenous nucleic acid encoding a methyl methacrylate pathway enzymeexpressed in a sufficient amount to produce a methyl methacrylate, themethyl methacrylate pathway comprising an alcohol transferase or anester-forming enzyme, and a dehydratase. In a further embodiment, themicrobial organism can comprise two exogenous nucleic acids eachencoding a methyl methacrylate pathway enzyme. In a particularembodiment, the two exogenous nucleic acids can encode an alcoholtransferase and a dehydratase or alternatively an ester-forming enzymeand a dehydratase. In another embodiment, the at least one exogenousnucleic acid can a heterologous nucleic acid. In another embodiment, thenon-naturally occurring microbial organism can be in a substantiallyanaerobic culture medium. The invention also provides a method forproducing methyl methacrylate by culturing a non-naturally occurringmicrobial organism having a methyl methacrylate pathway under conditionsand for a sufficient period of time to produce methyl methacrylate.

In one embodiment, the invention provides a non-naturally occurringmicrobial organism, comprising a microbial organism having amethacrylate ester pathway comprising at least one exogenous nucleicacid encoding a methacrylate ester pathway enzyme expressed in asufficient amount to produce a methacrylate ester, said methacrylateester pathway comprising an alcohol transferase or an ester-formingenzyme, and a dehydratase. In one aspect, the non-naturally occurringmicrobial organism comprises two exogenous nucleic acids each encoding amethacrylate ester pathway enzyme. In one aspect, the two exogenousnucleic acids encode an alcohol transferase and a dehydratase oralternatively an ester-forming enzyme and a dehydratase. In anotheraspect, the at least one exogenous nucleic acid is a heterologousnucleic acid. In another aspect, the non-naturally occurring microbialorganism is in a substantially anaerobic culture medium.

In one embodiment, the invention provides a non-naturally occurringmicrobial organism, wherein said dehydratase converts a3-hydroxyisobutyrate ester or a 2-hydroxyisobutyrate ester to saidmethacrylate ester. In one embodiment, the invention provides anon-naturally occurring microbial organism, wherein said alcoholtransferase coverts 3-hydroxyisobutyryl-CoA to a 3-hydroxyisobutyrateester or 2-hydroxyisobutyryl-CoA to 2-hydroxyisobutyrate ester.

In one embodiment, the invention provides a non-naturally occurringmicrobial organism, wherein said microbial organism further comprises amethacyrlate ester pathway comprising at least one exogenous nucleicacid encoding a methacrylate ester pathway enzyme expressed in asufficient amount to produce a methacrylate ester, said methacrylateester pathway comprising a pathway selected from: (a) a3-hydroxyisobutyrate-CoA transferase or a 3-hydroxyisobutyrate-CoAsynthetase; an alcohol transferase; and a dehydratase; (b) a3-hydroxyisobutyrate ester-forming enzyme and a dehydratasedehydratase;(c) a 2-hydroxyisobutyrate-CoA transferase or a 2-hydroxyisobutyrate-CoAsynthetase; an alcohol transferase; and a dehydratase; or (d) a2-hydroxyisobutyrate ester-forming enzyme and a dehydratase. In oneaspect, the invention provides a method for producing methacrylate esterby culturing the non-naturally occurring microbial organism as disclosedherein under conditions and for a sufficient period of time to producemethacrylate ester.

In one embodiment, the invention provides a non-naturally occurringmicrobial organism, comprising a microbial organism having a methylmethacrylate pathway comprising at least one exogenous nucleic acidencoding a methyl methacrylate pathway enzyme expressed in a sufficientamount to produce a methyl methacrylate, said methyl methacrylatepathway comprising an alcohol transferase or an ester-forming enzyme,and a dehydratase. In one aspect, the microbial organism comprises twoexogenous nucleic acids each encoding a methyl methacrylate pathwayenzyme. In another aspect, the two exogenous nucleic acids encode analcohol transferase and a dehydratase or alternatively an ester-formingenzyme and a dehydratase. In another aspect, the at least one exogenousnucleic acid is a heterologous nucleic acid. In another aspect, thenon-naturally occurring microbial organism is in a substantiallyanaerobic culture medium.

In one embodiment, the invention provides a method for producing methylmethacrylate, comprising culturing the non-naturally occurring microbialorganism as described herein under conditions and for a sufficientperiod of time to produce methyl methacrylate.

In one embodiment, the invention provides a method for producing amethacrylate ester comprising, culturing a non-naturally occurringmicrobial organism under conditions and for a sufficient period of timeto produce a 3-hydroxyisobutyrate ester, wherein said non-naturallyoccurring microbial organism comprises an exogenous nucleic acidencoding an alcohol transferase or a 3-hydroxyisobutyrate ester-formingenzyme expressed in a sufficient amount to produce a3-hydroxyisobutyrate ester, and chemically dehydrating said3-hydroxyisobutyrate ester to produce a methacrylate ester.

In one embodiment, the invention provides a method for producing amethacrylate ester comprising, culturing a non-naturally occurringmicrobial organism under conditions and for a sufficient period of timeto produce a 2-hydroxyisobutyrate ester, wherein said non-naturallyoccurring microbial organism comprises an exogenous nucleic acidencoding an alcohol transferase or a 2-hydroxyisobutyrate ester-formingenzyme expressed in a sufficient amount to produce a2-hydroxyisobutyrate ester, and chemically dehydrating said2-hydroxyisobutyrate ester to produce a methacrylate ester.

In one embodiment, the invention provides a method for producing methylmethacrylate comprising, culturing a non-naturally occurring microbialorganism under conditions and for a sufficient period of time to producemethyl-3-hydroxyisobutyrate, wherein said non-naturally occurringmicrobial organism comprises an exogenous nucleic acid encoding analcohol transferase or an ester-forming enzyme expressed in a sufficientamount to produce methyl-3-hydroxyisobutyrate, and chemicallydehydrating said methyl-3-hydroxyisobutyrate to produce methylmethacrylate.

In one aspect of the above methods, the exogenous nucleic acid is aheterologous nucleic acid. In another aspect of the above methods, thenon-naturally occurring microbial organism is in a substantiallyanaerobic culture medium.

In one embodiment, the invention provides a method wherein saidmicrobial organism further comprises a 3-hydroxyisobutyrate esterpathway comprising at least one exogenous nucleic acid encoding a3-hydroxyisobutyrate ester pathway enzyme expressed in a sufficientamount to produce a 3-hydroxyisobutyrate ester, said3-hydroxyisobutyrate ester pathway comprising a pathway selected from:(a) a 3-hydroxyisobutyrate-CoA transferase or a 3-hydroxyisobutyrate-CoAsynthetase; and an alcohol transferase; or (b) a 3-hydroxyisobutyrateester-forming enzyme. In another embodiment, the invention provides amethod wherein said microbial organism further comprises a2-hydroxyisobutyrate ester pathway comprising at least one exogenousnucleic acid encoding a 2-hydroxyisobutyrate ester pathway enzymeexpressed in a sufficient amount to produce a 2-hydroxyisobutyrateester, said 2-hydroxyisobutyrate ester pathway comprising a pathwayselected from: (a) a 2-hydroxyisobutyrate-CoA transferase or a2-hydroxyisobutyrate-CoA synthetase; and an alcohol transferase; or (b)a 2-hydroxyisobutyrate ester-forming enzyme.

As disclosed herein, the invention provides non-naturally occurringmicrobial organisms having a methacrylic acid pathway comprising atleast one exogenous nucleic acid encoding a methacrylic acid pathwayenzyme expressed in a sufficient amount to produce methacrylic acid.Exemplary pathways include, but are not limited to, the pathwaysdisclosed in FIG. 1. Exemplary methacrylic acid pathways include, forexample, the pathways corresponding to the enzymes shown in FIG. 1 asfollows and as discussed below in more detail: pathway (1) A/B/C;pathway (2) A/B/G/H; pathway (3) A/F/G/C; pathway (4) A/F/H; pathway (5)D/E/B/C; pathway (6) D/E/B/G/H; pathway (7) D/E/F/H; pathway (8)D/E/F/G/C; pathway (9) I/J/C; pathway (10) I/J/G/H; pathway (11)I/L/K/E/B/C; pathway (12) I/L/K/E/B/G/H; pathway (13) I/L/K/E/F/H; andpathway (14) I/L/K/E/F/G/C.

In a particular embodiment, the invention provides a non-naturallyoccurring microbial organism, comprising a microbial organism having amethacrylic acid pathway comprising at least one exogenous nucleic acidencoding a methacrylic acid pathway enzyme expressed in a sufficientamount to produce methacrylic acid, the methacrylic acid pathwaycomprising citramalate synthase (A), citramalate dehydratase(citraconate forming) (B), and citraconate decarboxylase (C) (FIG. 1,pathway (1) A/B/C). As disclosed herein, the microbial organism cancomprise more than one exogenous nucleic acid encoding a methacrylicacid pathway enzyme, including up to all enzymes in a pathway, forexample, three exogenous nucleic acids encode citramalate synthase,citramalate dehydratase (citraconate forming), and citraconatedecarboxylase.

In a particular embodiment, MAA producing non-naturally occurringmicrobial organism of the invention can have at least one exogenousnucleic acid that is a heterologous nucleic acid. In another embodimentthe non-naturally occurring microbial organism producing MAA can be in asubstantially anaerobic culture medium.

In another embodiment, the invention provides a non-naturally occurringmicrobial organism comprising a methacrylic acid pathway comprising atleast one exogenous nucleic acid encoding a methacrylic acid pathwayenzyme expressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising citramalate synthase (A),citramalate dehydratase (citraconate forming) (B), citraconate isomerase(G), and mesaconate decarboxylase (H) (FIG. 1, pathway (2) A/B/G/H). Instill another embodiment, the invention provides a non-naturallyoccurring microbial organism, comprising a methacrylic acid pathwaycomprising at least one exogenous nucleic acid encoding a methacrylicacid pathway enzyme expressed in a sufficient amount to producemethacrylic acid, the methacrylic acid pathway comprising citramalatesynthase (A), citramalate dehydratase (mesaconate forming) (F)citraconate isomerase (G), and citraconate decarboxylase (C) (FIG. 1,pathway (3) A/F/G/C).

In a further embodiment, the invention provides a non-naturallyoccurring microbial organism, comprising a methacrylic acid pathwaycomprising at least one exogenous nucleic acid encoding a methacrylicacid pathway enzyme expressed in a sufficient amount to producemethacrylic acid, the methacrylic acid pathway comprising citramalatesynthase (A), citramalate dehydratase (mesaconate forming) (F), andmesaconate decarboxylase (H) (FIG. 1, pathway (4) A/F/H). In yet afurther embodiment, the invention provides a non-naturally occurringmicrobial organism, comprising a methacrylic acid pathway comprising atleast one exogenous nucleic acid encoding a methacrylic acid pathwayenzyme expressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising citramalyl-CoA lyase (D),citramalyl-CoA transferase, synthetase or hydrolase (E), citramalatedehydratase (citraconate forming) (B), and citraconate decarboxylase (C)(FIG. 1, pathway (5) D/E/B/C).

In an additional embodiment, the invention provides a non-naturallyoccurring microbial organism, comprising a methacrylic acid pathwaycomprising at least one exogenous nucleic acid encoding a methacrylicacid pathway enzyme expressed in a sufficient amount to producemethacrylic acid, the methacrylic acid pathway comprising citramalyl-CoAlyase (D), citramalyl-CoA transferase, synthetase or hydrolase (E),citramalate dehydratase (citraconate forming) (B), citraconate isomerase(G), and mesaconate decarboxylase (H) (FIG. 1, pathway (6) D/E/B/G/H).In still another embodiment, the invention provides a non-naturallyoccurring microbial organism, comprising a methacrylic acid pathwaycomprising at least one exogenous nucleic acid encoding a methacrylicacid pathway enzyme expressed in a sufficient amount to producemethacrylic acid, the methacrylic acid pathway comprising citramalyl-CoAlyase (D), citramalyl-CoA transferase, synthetase or hydrolase (E),citramalate dehydratase (mesaconate forming) (F), and mesaconatedecarboxylase (H) (FIG. 1, pathway (7) D/E/F/H).

In another embodiment, the invention provides a non-naturally occurringmicrobial organism, comprising a methacrylic acid pathway comprising atleast one exogenous nucleic acid encoding a methacrylic acid pathwayenzyme expressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising citramalyl-CoA lyase (D),citramalyl-CoA transferase, synthetase or hydrolase (E), citramalatedehydratase (mesaconate forming) (F), citraconate isomerase (G), andcitraconate decarboxylase (C) (FIG. 1, pathway (8) D/E/F/G/C).Additionally, the invention provides a non-naturally occurring microbialorganism, comprising a methacrylic acid pathway comprising at least oneexogenous nucleic acid encoding a methacrylic acid pathway enzymeexpressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising aconitate decarboxylase (I),itaconate isomerase (J), and citraconate decarboxylase (C) (FIG. 1,pathway (9) I/J/C).

In yet a further embodiment, the invention provides a non-naturallyoccurring microbial organism, comprising a methacrylic acid pathwaycomprising at least one exogenous nucleic acid encoding a methacrylicacid pathway enzyme expressed in a sufficient amount to producemethacrylic acid, the methacrylic acid pathway comprising aconitatedecarboxylase (I), itaconate isomerase (J), citraconate isomerase (G),and mesaconate decarboxylase (H) (FIG. 1, pathway (10) I/J/G/H). In anadditional embodiment, the invention provides a non-naturally occurringmicrobial organism, comprising a methacrylic acid pathway comprising atleast one exogenous nucleic acid encoding a methacrylic acid pathwayenzyme expressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising aconitate decarboxylase (I),itaconyl-CoA transferase, synthetase or hydrolase (L), citramalyl-CoAdehydratase (K), citramalyl-CoA transferase, synthetase or hydrolase(E), citramalate dehydratase (citraconate forming) (B), and citraconatedecarboxylase (C) (FIG. 1, pathway (11) I/L/K/E/B/C).

The invention also provides a non-naturally occurring microbialorganism, comprising a methacrylic acid pathway comprising at least oneexogenous nucleic acid encoding a methacrylic acid pathway enzymeexpressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising aconitate decarboxylase (I),itaconyl-CoA transferase, synthetase or hydrolase (L), citramalyl-CoAdehydratase (K), citramalyl-CoA transferase, synthetase or hydrolase(E), citramalate dehydratase (citraconate forming) (B), citraconateisomerase (G), and mesaconate decarboxylase (H) (FIG. 1, pathway (12)I/L/K/E/B/G/H). In still a further embodiment, the invention provides anon-naturally occurring microbial organism, comprising a microbialorganism having a methacrylic acid pathway comprising at least oneexogenous nucleic acid encoding a methacrylic acid pathway enzymeexpressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising aconitate decarboxylase (I),itaconyl-CoA transferase, synthetase or hydrolase (L), citramalyl-CoAdehydratase (K), citramalyl-CoA transferase, synthetase or hydrolase(E), citramalate dehydratase (mesaconate forming) (F), and mesaconatedecarboxylase (H) (FIG. 1, pathway (13) I/L/K/E/F/H). In an additionalembodiment, the invention provides a non-naturally occurring microbialorganism, comprising a methacrylic acid pathway comprising at least oneexogenous nucleic acid encoding a methacrylic acid pathway enzymeexpressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising aconitate decarboxylase (I),itaconyl-CoA transferase, synthetase or hydrolase (L), citramalyl-CoAdehydratase (K), citramalyl-CoA transferase, synthetase or hydrolase(E), citramalate dehydratase (mesaconate forming) (F), citraconateisomerase (G), and citraconate decarboxylase (C) (FIG. 1, pathway (14)I/L/K/E/F/G/C).

In an additional embodiment, the invention provides a non-naturallyoccurring microbial organism having a methacrylic acid pathway, whereinthe non-naturally occurring microbial organism comprises at least oneexogenous nucleic acid encoding an enzyme or protein that converts asubstrate to a product selected from the group consisting of acetyl-CoAand pyruvate to citramalate, citramalate to citraconate, and citraconateto methacrylate; acetyl-CoA and pyruvate to citramalyl-CoA,citramalyl-CoA to citramalate, citramalate to citraconate, andcitraconate to methyacrylate; aconitate to itaconate, itaconate toitaconyl-CoA, itaconyl-CoA to citramalyl-CoA, citramalyl-CoA tocitramalate, citramalate to mesaconate, mesaconate to methacrylate, andso forth such as the reactions described herein and those substrates andproducts shown in the exemplary methacrylic acid pathways shown inFIG. 1. One skilled in the art will understand that these are merelyexemplary and that any of the substrate-product pairs disclosed hereinsuitable to produce a desired product and for which an appropriateactivity is available for the conversion of the substrate to the productcan be readily determined by one skilled in the art based on theteachings herein. Thus, the invention provides a non-naturally occurringmicrobial organism containing at least one exogenous nucleic acidencoding an enzyme or protein, where the enzyme or protein converts thesubstrates and products of a methacrylic acid pathway, such as thatshown in FIG. 1. Additionally provided is a methacrylic acid pathwaycomprising acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase,crotonase, 4-hydroxybutyryl-CoA dehydratase (or crotonyl-CoA hydratase,4-hydroxy), 4-hydroxybutyryl-CoA mutase, 3-hydroxyisobutyryl-CoAsynthetase or 3-hydroxyisobutyryl-CoA hydrolase or3-hydroxyisobutyryl-CoA transferase, and 3-hydroxyisobutyratedehydratase (see Example XXII and FIG. 21). Also provided is amethacrylic acid pathway comprising acetoacetyl-CoA thiolase,acetoacetyl-CoA reductase, crotonase, 4-hydroxybutyryl-CoA dehydratase,4-hydroxybutyryl-CoA mutase, 3-hydroxyisobutyryl-CoA dehydratase, andmethacrylyl-CoA synthetase or methacrylyl-CoA hydrolase ormethacrylyl-CoA transferase (see Example XXII and FIG. 21).

While generally described herein as a microbial organism that contains amethacrylic acid pathway, it is understood that the inventionadditionally provides a non-naturally occurring microbial organismcomprising at least one exogenous nucleic acid encoding a methacrylicacid pathway enzyme expressed in a sufficient amount to produce anintermediate of a methacrylic acid pathway. For example, as disclosedherein, a methacrylic acid pathway is exemplified in FIG. 1. Therefore,in addition to a microbial organism containing a methacrylic acidpathway that produces methacrylic acid, the invention additionallyprovides a non-naturally occurring microbial organism comprising atleast one exogenous nucleic acid encoding a methacrylic acid pathwayenzyme, where the microbial organism produces a methacrylic acid pathwayintermediate, for example, citramalyl-CoA, itaconyl-CoA, itaconate,citraconate, citramalate and mesaconate.

It is understood that any of the pathways disclosed herein, as describedin the Examples and exemplified in the pathways of FIGS. 1-30, can beutilized to generate a non-naturally occurring microbial organism thatproduces any pathway intermediate or product, as desired. As disclosedherein, such a microbial organism that produces an intermediate can beused in combination with another microbial organism expressingdownstream pathway enzymes to produce a desired product. However, it isunderstood that a non-naturally occurring microbial organism thatproduces a methacrylic acid pathway intermediate can be utilized toproduce the intermediate as a desired product.

This invention is also directed, in part to engineered biosyntheticpathways to improve carbon flux through a central metabolismintermediate en route to methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate. The present inventionprovides non-naturally occurring microbial organisms having one or moreexogenous genes encoding enzymes that can catalyze various enzymatictransformations en route to methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate. In some embodiments,these enzymatic transformations are part of the reductive tricarboxylicacid (RTCA) cycle and are used to improve product yields, including butnot limited to, from carbohydrate-based carbon feedstock.

In numerous engineered pathways, realization of maximum product yieldsbased on carbohydrate feedstock is hampered by insufficient reducingequivalents or by loss of reducing equivalents and/or carbon tobyproducts. In accordance with some embodiments, the present inventionincreases the yields of methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate by (i) enhancing carbonfixation via the reductive TCA cycle, and/or (ii) accessing additionalreducing equivalents from gaseous carbon sources and/or syngascomponents such as CO, CO₂, and/or H₂. In addition to syngas, othersources of such gases include, but are not limited to, the atmosphere,either as found in nature or generated.

The CO₂-fixing reductive tricarboxylic acid (RTCA) cycle is anendergenic anabolic pathway of CO₂ assimilation which uses reducingequivalents and ATP (FIG. 12). One turn of the RTCA cycle assimilatestwo moles of CO₂ into one mole of acetyl-CoA, or four moles of CO₂ intoone mole of oxaloacetate. This additional availability of acetyl-CoAimproves the maximum theoretical yield of product molecules derived fromcarbohydrate-based carbon feedstock. Exemplary carbohydrates include butare not limited to glucose, sucrose, xylose, arabinose and glycerol.

In some embodiments, the reductive TCA cycle, coupled with carbonmonoxide dehydrogenase and/or hydrogenase enzymes, can be employed toallow syngas, CO₂, CO, H₂, and/or other gaseous carbon sourceutilization by microorganisms. Synthesis gas (syngas), in particular isa mixture of primarily H₂ and CO, sometimes including some amounts ofCO₂, that can be obtained via gasification of any organic feedstock,such as coal, coal oil, natural gas, biomass, or waste organic matter.Numerous gasification processes have been developed, and most designsare based on partial oxidation, where limiting oxygen avoids fullcombustion, of organic materials at high temperatures (500-1500° C.) toprovide syngas as a 0.5:1-3:1 H₂/CO mixture. In addition to coal,biomass of many types has been used for syngas production and representsan inexpensive and flexible feedstock for the biological production ofrenewable chemicals and fuels. Carbon dioxide can be provided from theatmosphere or in condensed from, for example, from a tank cylinder, orvia sublimation of solid CO₂. Similarly, CO and hydrogen gas can beprovided in reagent form and/or mixed in any desired ratio. Othergaseous carbon forms can include, for example, methanol or similarvolatile organic solvents.

The components of synthesis gas and/or other carbon sources can providesufficient CO₂, reducing equivalents, and ATP for the reductive TCAcycle to operate. One turn of the RTCA cycle assimilates two moles ofCO₂ into one mole of acetyl-CoA and requires 2 ATP and 4 reducingequivalents. CO and/or H₂ can provide reducing equivalents by means ofcarbon monoxide dehydrogenase and hydrogenase enzymes, respectively.Reducing equivalents can come in the form of NADH, NADPH, FADH, reducedquinones, reduced ferredoxins, reduced flavodoxins and thioredoxins. Thereducing equivalents, particularly NADH, NADPH, and reduced ferredoxin,can serve as cofactors for the RTCA cycle enzymes, for example, malatedehydrogenase, fumarate reductase, alpha-ketoglutarate:ferredoxinoxidoreductase (alternatively known as 2-oxoglutarate:ferredoxinoxidoreductase, alpha-ketoglutarate synthase, or 2-oxoglutaratesynthase), pyruvate:ferredoxin oxidoreductase and isocitratedehydrogenase. The electrons from these reducing equivalents canalternatively pass through an ion-gradient producing electron transportchain where they are passed to an acceptor such as oxygen, nitrate,oxidized metal ions, protons, or an electrode. The ion-gradient can thenbe used for ATP generation via an ATP synthase or similar enzyme.

The reductive TCA cycle was first reported in the green sulfurphotosynthetic bacterium Chlorobium limicola (Evans et al., Proc. Natl.Acad. Sci. U.S.A. 55:928-934 (1966)). Similar pathways have beencharacterized in some prokaryotes (proteobacteria, green sulfur bacteriaand thermophilic Knallgas bacteria) and sulfur-dependent archaea (Hugleret al., J. Bacteriol. 187:3020-3027 (2005; Hugler et al., Environ.Microbiol. 9:81-92 (2007). In some cases, reductive and oxidative(Krebs) TCA cycles are present in the same organism (Hugler et al.,supra (2007); Siebers et al., J. Bacteriol. 186:2179-2194 (2004)). Somemethanogens and obligate anaerobes possess incomplete oxidative orreductive TCA cycles that may function to synthesize biosyntheticintermediates (Ekiel et al., J. Bacteriol. 162:905-908 (1985); Wood etal., FEMS Microbiol. Rev. 28:335-352 (2004)).

The key carbon-fixing enzymes of the reductive TCA cycle arealpha-ketoglutarate:ferredoxin oxidoreductase, pyruvate:ferredoxinoxidoreductase and isocitrate dehydrogenase. Additional carbon may befixed during the conversion of phosphoenolpyruvate to oxaloacetate byphosphoenolpyruvate carboxylase or phosphoenolpyruvate carboxykinase orby conversion of pyruvate to malate by malic enzyme.

Many of the enzymes in the TCA cycle are reversible and can catalyzereactions in the reductive and oxidative directions. However, some TCAcycle reactions are irreversible in vivo and thus different enzymes areused to catalyze these reactions in the directions required for thereverse TCA cycle. These reactions are: (1) conversion of citrate tooxaloacetate and acetyl-CoA, (2) conversion of fumarate to succinate,and (3) conversion of succinyl-CoA to alpha-ketoglutarate. In the TCAcycle, citrate is formed from the condensation of oxaloacetate andacetyl-CoA. The reverse reaction, cleavage of citrate to oxaloacetateand acetyl-CoA, is ATP-dependent and catalyzed by ATP-citrate lyase, orcitryl-CoA synthetase and citryl-CoA lyase. Alternatively, citrate lyasecan be coupled to acetyl-CoA synthetase, an acetyl-CoA transferase, orphosphotransacetylase and acetate kinase to form acetyl-CoA andoxaloacetate from citrate. The conversion of succinate to fumarate iscatalyzed by succinate dehydrogenase while the reverse reaction iscatalyzed by fumarate reductase. In the TCA cycle succinyl-CoA is formedfrom the NAD(P)⁺ dependent decarboxylation of alpha-ketoglutarate by thealpha-ketoglutarate dehydrogenase complex. The reverse reaction iscatalyzed by alpha-ketoglutarate:ferredoxin oxidoreductase.

An organism capable of utilizing the reverse tricarboxylic acid cycle toenable production of acetyl-CoA-derived products on 1) CO, 2) CO₂ andH₂, 3) CO and CO₂, 4) synthesis gas comprising CO and H₂, and 5)synthesis gas or other gaseous carbon sources comprising CO, CO₂, and H₂can include any of the following enzyme activities: ATP-citrate lyase,citrate lyase, aconitase, isocitrate dehydrogenase,alpha-ketoglutarate:ferredoxin oxidoreductase, succinyl-CoA synthetase,succinyl-CoA transferase, fumarate reductase, fumarase, malatedehydrogenase, acetate kinase, phosphotransacetylase, acetyl-CoAsynthetase, acetyl-CoA transferase, pyruvate:ferredoxin oxidoreductase,NAD(P)H:ferredoxin oxidoreductase, carbon monoxide dehydrogenase,hydrogenase, and ferredoxin (see FIG. 13). Enzyme enzymes and thecorresponding genes required for these activities are described herein.

Carbon from syngas or other gaseous carbon sources can be fixed via thereverse TCA cycle and components thereof. Specifically, the combinationof certain carbon gas-utilization pathway components with the pathwaysfor formation of methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate from acetyl-CoA resultsin high yields of these products by providing an efficient mechanism forfixing the carbon present in carbon dioxide, fed exogenously or producedendogenously from CO, into acetyl-CoA.

In some embodiments, a methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate pathway in anon-naturally occurring microbial organism of the invention can utilizeany combination of (1) CO, (2) CO₂, (3) H₂, or mixtures thereof toenhance the yields of biosynthetic steps involving reduction, includingaddition to driving the reductive TCA cycle.

In some embodiments a non-naturally occurring microbial organism havingan methacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate pathway includes at least one exogenous nucleicacid encoding a reductive TCA pathway enzyme. The at least one exogenousnucleic acid is selected from an ATP-citrate lyase, citrate lyase, afumarate reductase, isocitrate dehydrogenase, aconitase, and analpha-ketoglutarate:ferredoxin oxidoreductase; and at least oneexogenous enzyme selected from a carbon monoxide dehydrogenase, ahydrogenase, a NAD(P)H:ferredoxin oxidoreductase, and a ferredoxin,expressed in a sufficient amount to allow the utilization of (1) CO, (2)CO₂, (3) H₂, (4) CO₂ and H₂, (5) CO and CO₂, (6) CO and H₂, or (7) CO,CO₂, and H₂.

In some embodiments a method includes culturing a non-naturallyoccurring microbial organism having a methacrylic acid, methacrylateester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate pathway alsocomprising at least one exogenous nucleic acid encoding a reductive TCApathway enzyme. The at least one exogenous nucleic acid is selected froman ATP-citrate lyase, citrate lyase, a fumarate reductase, isocitratedehydrogenase, aconitase, and an alpha-ketoglutarate:ferredoxinoxidoreductase. Additionally, such an organism can also include at leastone exogenous enzyme selected from a carbon monoxide dehydrogenase, ahydrogenase, a NAD(P)H:ferredoxin oxidoreductase, and a ferredoxin,expressed in a sufficient amount to allow the utilization of (1) CO, (2)CO₂, (3) H₂, (4) CO₂ and H₂, (5) CO and CO₂, (6) CO and H₂, or (7) CO,CO₂, and H₂ to produce a product.

In some embodiments a non-naturally occurring microbial organism havingan methacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate pathway further includes at least one exogenousnucleic acid encoding a reductive TCA pathway enzyme expressed in asufficient amount to enhance carbon flux through acetyl-CoA. The atleast one exogenous nucleic acid is selected from an ATP-citrate lyase,citrate lyase, a fumarate reductase, a pyruvate:ferredoxinoxidoreductase, isocitrate dehydrogenase, aconitase and analpha-ketoglutarate:ferredoxin oxidoreductase.

In some embodiments a non-naturally occurring microbial organism havingan methacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate pathway includes at least one exogenous nucleicacid encoding an enzyme expressed in a sufficient amount to enhance theavailability of reducing equivalents in the presence of carbon monoxideand/or hydrogen, thereby increasing the yield of redox-limited productsvia carbohydrate-based carbon feedstock. The at least one exogenousnucleic acid is selected from a carbon monoxide dehydrogenase, ahydrogenase, an NAD(P)H:ferredoxin oxidoreductase, and a ferredoxin. Insome embodiments, the present invention provides a method for enhancingthe availability of reducing equivalents in the presence of carbonmonoxide or hydrogen thereby increasing the yield of redox-limitedproducts via carbohydrate-based carbon feedstock, such as sugars orgaseous carbon sources, the method includes culturing this non-naturallyoccurring microbial organism under conditions and for a sufficientperiod of time to produce methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate.

In some embodiments, the non-naturally occurring microbial organismhaving an methacrylic acid, methacrylate ester, 3-hydroxyisobutyrateand/or 2-hydroxyisobutyrate pathway includes two exogenous nucleic acidseach encoding a reductive TCA pathway enzyme. In some embodiments, thenon-naturally occurring microbial organism having an methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyratepathway includes three exogenous nucleic acids each encoding a reductiveTCA pathway enzyme. In some embodiments, the non-naturally occurringmicrobial organism includes three exogenous nucleic acids encoding anATP-citrate lyase, a fumarate reductase, and analpha-ketoglutarate:ferredoxin oxidoreductase. In some embodiments, thenon-naturally occurring microbial organism includes three exogenousnucleic acids encoding a citrate lyase, a fumarate reductase, and analpha-ketoglutarate:ferredoxin oxidoreductase. In some embodiments, thenon-naturally occurring microbial organism includes four exogenousnucleic acids encoding a pyruvate:ferredoxin oxidoreductase; aphosphoenolpyruvate carboxylase or a phosphoenolpyruvate carboxykinase,a CO dehydrogenase; and an H₂ hydrogenase. In some embodiments, thenon-naturally occurring microbial organism includes two exogenousnucleic acids encoding a CO dehydrogenase and an H₂ hydrogenase.

In some embodiments, the non-naturally occurring microbial organismshaving an methacrylic acid, methacrylate ester, 3-hydroxyisobutyrateand/or 2-hydroxyisobutyrate pathway further include an exogenous nucleicacid encoding an enzyme selected from a pyruvate:ferredoxinoxidoreductase, an aconitase, an isocitrate dehydrogenase, asuccinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, amalate dehydrogenase, an acetate kinase, a phosphotransacetylase, anacetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, andcombinations thereof.

In some embodiments, the non-naturally occurring microbial organismhaving an methacrylic acid, methacrylate ester, 3-hydroxyisobutyrateand/or 2-hydroxyisobutyrate pathway further includes an exogenousnucleic acid encoding an enzyme selected from carbon monoxidedehydrogenase, acetyl-CoA synthase, ferredoxin, NAD(P)H:ferredoxinoxidoreductase and combinations thereof.

In some embodiments, the non-naturally occurring microbial organismhaving an methacrylic acid, methacrylate ester, 3-hydroxyisobutyrateand/or 2-hydroxyisobutyrate pathway utilizes a carbon feedstock selectedfrom (1) CO, (2) CO₂, (3) CO₂ and H₂, (4) CO and H₂, or (5) CO, CO₂, andH₂. In some embodiments, the non-naturally occurring microbial organismhaving an methacrylic acid, methacrylate ester, 3-hydroxyisobutyrateand/or 2-hydroxyisobutyrate pathway utilizes hydrogen for reducingequivalents. In some embodiments, the non-naturally occurring microbialorganism having an methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate pathway utilizes CO forreducing equivalents. In some embodiments, the non-naturally occurringmicrobial organism having an methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate pathway utilizescombinations of CO and hydrogen for reducing equivalents.

In some embodiments, the non-naturally occurring microbial organismhaving an methacrylic acid, methacrylate ester, 3-hydroxyisobutyrateand/or 2-hydroxyisobutyrate pathway further includes one or more nucleicacids encoding an enzyme selected from a phosphoenolpyruvatecarboxylase, a phosphoenolpyruvate carboxykinase, a pyruvatecarboxylase, and a malic enzyme.

In some embodiments, the non-naturally occurring microbial organismhaving an methacrylic acid, methacrylate ester, 3-hydroxyisobutyrateand/or 2-hydroxyisobutyrate pathway further includes one or more nucleicacids encoding an enzyme selected from a malate dehydrogenase, afumarase, a fumarate reductase, a succinyl-CoA synthetase, and asuccinyl-CoA transferase.

In some embodiments, the non-naturally occurring microbial organismhaving an methacrylic acid, methacrylate ester, 3-hydroxyisobutyrateand/or 2-hydroxyisobutyrate pathway can have or optionally furtherincludes at least one exogenous nucleic acid encoding a citrate lyase,an ATP-citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase anaconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, asuccinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetatekinase, a phosphotransacetylase, an acetyl-CoA synthetase, and aferredoxin.

It is understood by those skilled in the art that the pathways describedherein for increasing product yield can be combined with any of thepathways disclosed herein, including those pathways depicted in thefigures. One skilled in the art will understand that, depending on thepathway to a desired product and the precursors and intermediates ofthat pathway, a particular pathway for improving product yield, asdiscussed herein and in the examples, or combination of such pathways,can be used in combination with a pathway to a desired product toincrease the yield of that product or a pathway intermediate.

The invention also provides a non-naturally occurring microbialorganism, comprising a microbial organism having a methacrylic acidpathway comprising at least one exogenous nucleic acid encoding amethacrylic acid pathway enzyme expressed in a sufficient amount toproduce methacrylic acid; said non-naturally occurring microbialorganism further comprising: (i) a reductive TCA pathway comprising atleast one exogenous nucleic acid encoding a reductive TCA pathwayenzyme, wherein said at least one exogenous nucleic acid is selectedfrom an ATP-citrate lyase, citrate lyase, a fumarate reductase, and analpha-ketoglutarate:ferredoxin oxidoreductase, or optionally isocitratedehydrogenase, aconitase, citryl-CoA synthetase or citryl-CoA lyase;(ii) a reductive TCA pathway comprising at least one exogenous nucleicacid encoding a reductive TCA pathway enzyme, wherein said at least oneexogenous nucleic acid is selected from a pyruvate:ferredoxinoxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvatecarboxykinase, a CO dehydrogenase, and an H₂ hydrogenase; or (iii) atleast one exogenous nucleic acid encodes an enzyme selected from a COdehydrogenase, an H₂ hydrogenase, and combinations thereof; wherein saidmethacrylic acid pathway comprises a pathway selected from: (a)citramalate synthase, citramalate dehydratase (citraconate forming), andcitraconate decarboxylase; (b) citramalate synthase, citramalatedehydratase (citraconate forming), citraconate isomerase, and mesaconatedecarboxylase; (c) citramalate synthase, citramalate dehydratase(mesaconate forming), citraconate isomerase, and citraconatedecarboxylase; (d) citramalate synthase, citramalate dehydratase(mesaconate forming), and mesaconate decarboxylase; (e) citramalyl-CoAlyase, citramalyl-CoA transferase, synthetase or hydrolase, citramalatedehydratase (citraconate forming), and citraconate decarboxylase; (f)citramalyl-CoA lyase, citramalyl-CoA transferase, synthetase orhydrolase, citramalate dehydratase (citraconate forming), citraconateisomerase, and mesaconate decarboxylase; (g) citramalyl-CoA lyase,citramalyl-CoA transferase, synthetase or hydrolase, citramalatedehydratase (mesaconate forming), and mesaconate decarboxylase; (h)citramalyl-CoA lyase, citramalyl-CoA transferase, synthetase orhydrolase, citramalate dehydratase (mesaconate forming), citraconateisomerase, and citraconate decarboxylase; (i) aconitate decarboxylase,itaconate isomerase, and citraconate decarboxylase; (j) aconitatedecarboxylase, itaconate isomerase, citraconate isomerase, andmesaconate decarboxylase; (k) aconitate decarboxylase, itaconyl-CoAtransferase, synthetase or hydrolase, citramalyl-CoA dehydratase,citramalyl-CoA transferase, synthetase or hydrolase, citramalatedehydratase (citraconate forming), and citraconate decarboxylase; (l)aconitate decarboxylase, itaconyl-CoA transferase, synthetase orhydrolase, citramalyl-CoA dehydratase, citramalyl-CoA transferase,synthetase or hydrolase, citramalate dehydratase (citraconate forming),citraconate isomerase, and mesaconate decarboxylase; (m) aconitatedecarboxylase, itaconyl-CoA transferase, synthetase or hydrolase,citramalyl-CoA dehydratase, citramalyl-CoA transferase, synthetase orhydrolase, citramalate dehydratase (mesaconate forming), and mesaconatedecarboxylase; (n) aconitate decarboxylase, itaconyl-CoA transferase,synthetase or hydrolase, citramalyl-CoA dehydratase, citramalyl-CoAtransferase, synthetase or hydrolase, citramalate dehydratase(mesaconate forming), citraconate isomerase, and citraconatedecarboxylase; (o) 3-hydroxyisobutyrate dehydratase; (p)methylmalonyl-CoA mutase, methylmalonyl-CoA reductase,3-hydroxyisobutyrate dehydrogenase, and 3-hydroxyisobutyratedehydratase; (q) methylmalonyl-CoA mutase, methylmalonyl-CoA epimerase,methylmalonyl-CoA reductase, 3-hydroxyisobutyrate dehydrogenase, and3-hydroxyisobutyrate dehydratase; (r) methylmalonyl-CoA mutase,alcohol/aldehyde dehydrogenase, and 3-hydroxyisobutyrate dehydratase;(s) methylmalonyl-CoA mutase, methylmalonyl-CoA epimerase,alcohol/aldehyde dehydrogenase, and 3-hydroxyisobutyrate dehydratase;(t) methylmalonyl-CoA mutase, methylmalonyl-CoA reductase,3-amino-2-methylpropionate transaminase, and 3-amino-2-methylpropionateammonia lyase; (u) methylmalonyl-CoA mutase, methylmalonyl-CoAepimerase, methylmalonyl-CoA reductase, 3-amino-2-methylpropionatetransaminase, and 3-amino-2-methylpropionate ammonia lyase; (v)4-hydroxybutyryl-CoA mutase, 3-hydroxyisobutyryl-CoA synthetase or3-hydroxyisobutyryl-CoA hydrolase or 3-hydroxyisobutyryl-CoAtransferase, and 3-hydroxyisobutyrate dehydratase; (w) aspartateaminotransferase, glutamate mutase, 3-methylaspartase, and mesaconatedecarboxylase; (x) alpha-ketoglutarate reductase, 2-hydroxyglutamatemutase, 3-methylmalate dehydratase, and mesaconate decarboxylase; (y)acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase,3-hydroxybutyryl-CoA mutase, 2-hydroxyisobutyryl-CoA dehydratase, andmethacrylyl-CoA transferase or methacrylyl-CoA hydrolase ormethacrylyl-CoA synthetase; (z) acetoacetyl-CoA thiolase,acetoacetyl-CoA reductase, 3-hydroxybutyryl-CoA mutase,2-hydroxyisobutyryl-CoA dehydratase, enoyl-CoA hydratase, and3-hydroxyisobutyryl-CoA hydrolase or 3-hydroxyisobutyryl-CoA synthetaseor 3-hydroxyisobutyryl-CoA transferase, and 3-hydroxyisobutyratedehydratase; (aa) 4-hydroxybutyryl-CoA dehydratase, vinylacetyl-CoAΔ-isomerase, crotonase, 3-hydroxybutyryl-CoA mutase,2-hydroxyisobutyryl-CoA dehydratase, and any of methacrylyl-CoAhydrolase or methacrylyl-CoA synthetase or methacrylyl-CoA transferase;(bb) 4-hydroxybutyryl-CoA mutase, 3-hydroxyisobutyryl-CoA dehydratase,and methacrylyl-CoA synthetase or methacrylyl-CoA hydrolase ormethacrylyl-CoA transferase; (cc) acetoacetyl-CoA thiolase,acetoacetyl-CoA reductase, crotonase, butyryl-CoA dehydrogenase,isobutyryl-CoA mutase, isobutyryl-CoA dehydrogenase, and methacrylyl-CoAsynthetase or methacrylyl-CoA hydrolase or methacrylyl-CoA transferase;(dd) lactate dehydrogenase, lactate-CoA transferase, lactoyl-CoAdehydratase, acyl-CoA dehydrogenase, propionyl-CoA carboxylase,methylmalonyl-CoA reductase, 3-hydroxyisobutyrate dehydrogenase, and3-hydroxyisobutyrate dehydratase; (ee) valine aminotransferase,2-ketoisovalerate dehydrogenase, isobutyryl-CoA dehydrogenase, andmethacrylyl-CoA synthetase or methacrylyl-CoA hydrolase ormethacrylyl-CoA transferase; (ff) valine aminotransferase,2-ketoisovalerate dehydrogenase, isobutyryl-CoA dehydrogenase,methacrylyl-CoA synthetase or methacrylyl-CoA hydrolase ormethacrylyl-CoA transferase, acetolactate synthase, acetohydroxy acidisomeroreductase and dihydroxy-acid dehydratase; (gg) acetoacetyl-CoAthiolase, acetoacetyl-CoA reductase, crotonase, 4-hydroxybutyryl-CoAdehydratase, 4-hydroxybutyryl-CoA mutase, 3-hydroxyisobutyryl-CoAsynthetase or 3-hydroxyisobutyryl-CoA hydrolase or3-hydroxyisobutyryl-CoA transferase, and 3-hydroxyisobutyratedehydratase; and (hh) acetoacetyl-CoA thiolase, acetoacetyl-CoAreductase, crotonase, 4-hydroxybutyryl-CoA dehydratase,4-hydroxybutyryl-CoA mutase, 3-hydroxyisobutyryl-CoA dehydratase, andmethacrylyl-CoA synthetase or methacrylyl-CoA hydrolase ormethacrylyl-CoA transferase.

The invention additionally provides a non-naturally occurring microbialorganism having a 2-hydroxyisobutyric acid pathway comprising at leastone exogenous nucleic acid encoding a 2-hydroxyisobutyric acid pathwayenzyme expressed in a sufficient amount to produce 2-hydroxyisobutyricacid. Such a non-naturally occurring microbial organism can furthercomprise (i) a reductive TCA pathway comprising at least one exogenousnucleic acid encoding a reductive TCA pathway enzyme, wherein the atleast one exogenous nucleic acid is selected from an ATP-citrate lyase,citrate lyase, a fumarate reductase, and analpha-ketoglutarate:ferredoxin oxidoreductase, or optionally isocitratedehydrogenase, aconitase, citryl-CoA synthetase or citryl-CoA lyase;(ii) a reductive TCA pathway comprising at least one exogenous nucleicacid encoding a reductive TCA pathway enzyme, wherein the at least oneexogenous nucleic acid is selected from a pyruvate:ferredoxinoxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvatecarboxykinase, a CO dehydrogenase, and an H₂ hydrogenase; or (iii) atleast one exogenous nucleic acid encodes an enzyme selected from a COdehydrogenase, an H₂ hydrogenase, and combinations thereof; wherein the2-hydroxyisobutyric acid pathway comprises a pathway selected from (a)acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase,3-hydroxybutyryl-CoA mutase, and 2-hydroxyisobutyryl-CoA transferase or2-hydroxyisobutyryl-CoA hydrolase or 2-hydroxyisobutyryl-CoA synthetase;and (b) 4-hydroxybutyryl-CoA dehydratase, vinylacetyl-CoA Δ-isomerase,crotonase, 3-hydroxybutyryl-CoA mutase, and any of2-hydroxyisobutyryl-CoA hydrolase or 2-hydroxyisobutyryl-CoA synthetaseor 2-hydroxyisobutyryl-CoA transferase (see FIG. 16C). The non-naturallyoccurring microbial organism can comprise two, three, four or fiveexogenous nucleic acids each encoding a methacrylic acid pathway enzyme,up to all enzymes of the pathway.

The invention further provides a non-naturally occurring microbialorganism comprising a microbial organism having a 3-hydroxyisobutyricacid pathway comprising at least one exogenous nucleic acid encoding a3-hydroxyisobutyric acid pathway enzyme expressed in a sufficient amountto produce 3-hydroxyisobutyric acid. The non-naturally occurringmicrobial organism can further comprise (i) a reductive TCA pathwaycomprising at least one exogenous nucleic acid encoding a reductive TCApathway enzyme, wherein said at least one exogenous nucleic acid isselected from an ATP-citrate lyase, citrate lyase, a fumarate reductase,and an alpha-ketoglutarate:ferredoxin oxidoreductase, or optionallyisocitrate dehydrogenase, aconitase, citryl-CoA synthetase or citryl-CoAlyase; (ii) a reductive TCA pathway comprising at least one exogenousnucleic acid encoding a reductive TCA pathway enzyme, wherein the atleast one exogenous nucleic acid is selected from a pyruvate:ferredoxinoxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvatecarboxykinase, a CO dehydrogenase, and an H₂ hydrogenase; or (iii) atleast one exogenous nucleic acid encodes an enzyme selected from a COdehydrogenase, an H₂ hydrogenase, and combinations thereof; wherein the3-hydroxyisobutyric acid pathway comprises a pathway selected from (a)4-hydroxybutyryl-CoA mutase; and (b) 4-hydroxybutyryl-CoA mutase and3-hydroxyisobutyryl-CoA synthetase or 3-hydroxyisobutyryl-CoA hydrolaseor 3-hydroxyisobutyryl-CoA transferase (see FIG. 16B). The non-naturallyoccurring microbial organism can comprise two exogenous nucleic acidseach encoding a methacrylic acid pathway enzyme.

The invention also provides a non-naturally occurring microbial organismcomprising a microbial organism having a 3-hydroxyisobutyryl-CoA pathwaycomprising at least one exogenous nucleic acid encoding a3-hydroxyisobutyric acid pathway enzyme expressed in a sufficient amountto produce 3-hydroxyisobutyric acid. The non-naturally occurringmicrobial organism can further comprise (i) a reductive TCA pathwaycomprising at least one exogenous nucleic acid encoding a reductive TCApathway enzyme, wherein the at least one exogenous nucleic acid isselected from an ATP-citrate lyase, citrate lyase, a fumarate reductase,and an alpha-ketoglutarate:ferredoxin oxidoreductase, or optionallyisocitrate dehydrogenase, aconitase, citryl-CoA synthetase or citryl-CoAlyase; (ii) a reductive TCA pathway comprising at least one exogenousnucleic acid encoding a reductive TCA pathway enzyme, wherein the atleast one exogenous nucleic acid is selected from a pyruvate:ferredoxinoxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvatecarboxykinase, a CO dehydrogenase, and an H₂ hydrogenase; or (iii) atleast one exogenous nucleic acid encodes an enzyme selected from a COdehydrogenase, an H₂ hydrogenase, and combinations thereof; wherein the3-hydroxyisobutyryl-CoA pathway comprises 4-hydroxybutyryl-CoA mutase(see FIG. 16B).

In a further embodiment, the microbial organism comprising (i) furthercomprises an exogenous nucleic acid encoding an enzyme selected from apyruvate:ferredoxin oxidoreductase, an aconitase, an isocitratedehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, afumarase, a malate dehydrogenase, an acetate kinase, aphosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxinoxidoreductase, ferredoxin, and combinations thereof; a microbialorganism comprising (ii) further comprises an exogenous nucleic acidencoding an enzyme selected from an aconitase, an isocitratedehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, afumarase, a malate dehydrogenase, and combinations thereof.

Such a microbial organism can comprise two, three, four, five, six orseven exogenous nucleic acids each encoding a methacrylic acid pathwayenzyme, up to all of the enzymes of a pathway. For example, themicrobial organism can comprises (a) three exogenous nucleic acidsencoding citramalate synthase, citramalate dehydratase (citraconateforming), and citraconate decarboxylase; (b) four exogenous nucleicacids encoding citramalate synthase, citramalate dehydratase(citraconate forming), citraconate isomerase, and mesaconatedecarboxylase; (c) four exogenous nucleic acids encoding citramalatesynthase, citramalate dehydratase (mesaconate forming), citraconateisomerase, and citraconate decarboxylase; (d) three exogenous nucleicacids encoding citramalate synthase, citramalate dehydratase (mesaconateforming), and mesaconate decarboxylase; (e) four exogenous nucleic acidsencoding citramalyl-CoA lyase, citramalyl-CoA transferase, synthetase orhydrolase, citramalate dehydratase (citraconate forming), andcitraconate decarboxylase; (f) five exogenous nucleic acids encodingcitramalyl-CoA lyase, citramalyl-CoA transferase, synthetase orhydrolase, citramalate dehydratase (citraconate forming), citraconateisomerase, and mesaconate decarboxylase; (g) four exogenous nucleicacids encoding citramalyl-CoA lyase, citramalyl-CoA transferase,synthetase or hydrolase, citramalate dehydratase (mesaconate forming),and mesaconate decarboxylase; (h) five exogenous nucleic acids encodingcitramalyl-CoA lyase, citramalyl-CoA transferase, synthetase orhydrolase, citramalate dehydratase (mesaconate forming), citraconateisomerase, and citraconate decarboxylase; (i) three exogenous nucleicacids encoding aconitate decarboxylase, itaconate isomerase, andcitraconate decarboxylase; (j) four exogenous nucleic acids encodingaconitate decarboxylase, itaconate isomerase, citraconate isomerase, andmesaconate decarboxylase; (k) six exogenous nucleic acids encodingaconitate decarboxylase, itaconyl-CoA transferase, synthetase orhydrolase, citramalyl-CoA dehydratase, citramalyl-CoA transferase,synthetase or hydrolase, citramalate dehydratase (citraconate forming),and citraconate decarboxylase; (l) seven exogenous nucleic acidsencoding aconitate decarboxylase, itaconyl-CoA transferase, synthetaseor hydrolase, citramalyl-CoA dehydratase, citramalyl-CoA transferase,synthetase or hydrolase, citramalate dehydratase (citraconate forming),citraconate isomerase, and mesaconate decarboxylase; (m) six exogenousnucleic acids encoding aconitate decarboxylase, itaconyl-CoAtransferase, synthetase or hydrolase, citramalyl-CoA dehydratase,citramalyl-CoA transferase, synthetase or hydrolase, citramalatedehydratase (mesaconate forming), and mesaconate decarboxylase; or (n)seven exogenous nucleic acids encoding aconitate decarboxylase,itaconyl-CoA transferase, synthetase or hydrolase, citramalyl-CoAdehydratase, citramalyl-CoA transferase, synthetase or hydrolase,citramalate dehydratase (mesaconate forming), citraconate isomerase, andcitraconate decarboxylase; (o) one exogenous nucleic acid encoding3-hydroxyisobutyrate dehydratase; (p) four exogenous nucleic acidsencoding methylmalonyl-CoA mutase, methylmalonyl-CoA reductase,3-hydroxyisobutyrate dehydrogenase, and 3-hydroxyisobutyratedehydratase; (q) five exogenous nucleic acids encoding methylmalonyl-CoAmutase, methylmalonyl-CoA epimerase, methylmalonyl-CoA reductase,3-hydroxyisobutyrate dehydrogenase, and 3-hydroxyisobutyratedehydratase; (r) three exogenous nucleic acids encodingmethylmalonyl-CoA mutase, alcohol/aldehyde dehydrogenase, and3-hydroxyisobutyrate dehydratase; (s) four exogenous nucleic acidsencoding methylmalonyl-CoA mutase, methylmalonyl-CoA epimerase,alcohol/aldehyde dehydrogenase, and 3-hydroxyisobutyrate dehydratase;(t) four exogenous nucleic acids encoding methylmalonyl-CoA mutase,methylmalonyl-CoA reductase, 3-amino-2-methylpropionate transaminase,and 3-amino-2-methylpropionate ammonia lyase; (u) five exogenous nucleicacids encoding methylmalonyl-CoA mutase, methylmalonyl-CoA epimerase,methylmalonyl-CoA reductase, 3-amino-2-methylpropionate transaminase,and 3-amino-2-methylpropionate ammonia lyase; (v) three exogenousnucleic acids encoding 4-hydroxybutyryl-CoA mutase,3-hydroxyisobutyryl-CoA synthetase or 3-hydroxyisobutyryl-CoA hydrolaseor 3-hydroxyisobutyryl-CoA transferase, and 3-hydroxyisobutyratedehydratase; (w) four exogenous nucleic acids encoding aspartateaminotransferase, glutamate mutase, 3-methylaspartase, and mesaconatedecarboxylase; (x) four exogenous nucleic acids encodingalpha-ketoglutarate reductase, 2-hydroxyglutamate mutase, 3-methylmalatedehydratase, and mesaconate decarboxylase; (y) five exogenous nucleicacids encoding acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase,3-hydroxybutyryl-CoA mutase, 2-hydroxyisobutyryl-CoA dehydratase, andmethacrylyl-CoA transferase or methacrylyl-CoA hydrolase ormethacrylyl-CoA synthetase; (z) seven exogenous nucleic acids encodingacetoacetyl-CoA thiolase, acetoacetyl-CoA reductase,3-hydroxybutyryl-CoA mutase, 2-hydroxyisobutyryl-CoA dehydratase,enoyl-CoA hydratase, and 3-hydroxyisobutyryl-CoA hydrolase or3-hydroxyisobutyryl-CoA synthetase or 3-hydroxyisobutyryl-CoAtransferase, and 3-hydroxyisobutyrate dehydratase; (aa) six exogenousnucleic acids encoding 4-hydroxybutyryl-CoA dehydratase, vinylacetyl-CoAΔ-isomerase, crotonase, 3-hydroxybutyryl-CoA mutase,2-hydroxyisobutyryl-CoA dehydratase, and any of methacrylyl-CoAhydrolase or methacrylyl-CoA synthetase or methacrylyl-CoA transferase;(bb) three exogenous nucleic acids encoding 4-hydroxybutyryl-CoA mutase,3-hydroxyisobutyryl-CoA dehydratase, and methacrylyl-CoA synthetase ormethacrylyl-CoA hydrolase or methacrylyl-CoA transferase; (cc)acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, crotonase,butyryl-CoA dehydrogenase, isobutyryl-CoA mutase, isobutyryl-CoAdehydrogenase, and methacrylyl-CoA synthetase or methacrylyl-CoAhydrolase or methacrylyl-CoA transferase; (dd) lactate dehydrogenase,lactate-CoA transferase, lactoyl-CoA dehydratase, acyl-CoAdehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA reductase,3-hydroxyisobutyrate dehydrogenase, and 3-hydroxyisobutyratedehydratase; (ee) valine aminotransferase, 2-ketoisovaleratedehydrogenase, isobutyryl-CoA dehydrogenase, and methacrylyl-CoAsynthetase or methacrylyl-CoA hydrolase or methacrylyl-CoA transferase;(ff) valine aminotransferase, 2-ketoisovalerate dehydrogenase,isobutyryl-CoA dehydrogenase, methacrylyl-CoA synthetase ormethacrylyl-CoA hydrolase or methacrylyl-CoA transferase, acetolactatesynthase, acetohydroxy acid isomeroreductase and dihydroxy-aciddehydratase; (gg) acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase,crotonase, 4-hydroxybutyryl-CoA dehydratase, 4-hydroxybutyryl-CoAmutase, 3-hydroxyisobutyryl-CoA synthetase or 3-hydroxyisobutyryl-CoAhydrolase or 3-hydroxyisobutyryl-CoA transferase, and3-hydroxyisobutyrate dehydratase; and (hh) acetoacetyl-CoA thiolase,acetoacetyl-CoA reductase, crotonase, 4-hydroxybutyryl-CoA dehydratase,4-hydroxybutyryl-CoA mutase, 3-hydroxyisobutyryl-CoA dehydratase, andmethacrylyl-CoA synthetase or methacrylyl-CoA hydrolase ormethacrylyl-CoA transferase.

In another embodiment, a microbial organism can comprise two, three,four or five exogenous nucleic acids each encoding enzymes of (i), (ii)or (iii). For example, amicrobial organism comprising (i) can comprisefour exogenous nucleic acids encoding ATP-citrate lyase, citrate lyase,a fumarate reductase, and an alpha-ketoglutarate:ferredoxinoxidoreductase; amicrobial organism comprising (ii) can comprise fiveexogenous nucleic acids encoding pyruvate:ferredoxin oxidoreductase, aphosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, aCO dehydrogenase, and an H₂ hydrogenase; or a microbial organismcomprising (iii) can comprise two exogenous nucleic acids encoding COdehydrogenase and H₂ hydrogenase.

The invention additionally provides a non-naturally occurring microbialorganism comprising a microbial organism having a 2-hydroxyisobutyricacid pathway comprising at least one exogenous nucleic acid encoding a2-hydroxyisobutyric acid pathway enzyme expressed in a sufficient amountto produce 2-hydroxyisobutyric acid; wherein said 2-hydroxyisobutyricacid pathway enzymes are selected from those shown in FIG. 16C; saidnon-naturally occurring microbial organism further comprising: (i) areductive TCA pathway comprising at least one exogenous nucleic acidencoding a reductive TCA pathway enzyme, wherein said at least oneexogenous nucleic acid is selected from an ATP-citrate lyase, citratelyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxinoxidoreductase, or optionally isocitrate dehydrogenase, aconitase,citryl-CoA synthetase or citryl-CoA lyase; (ii) a reductive TCA pathwaycomprising at least one exogenous nucleic acid encoding a reductive TCApathway enzyme, wherein said at least one exogenous nucleic acid isselected from a pyruvate:ferredoxin oxidoreductase, aphosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, aCO dehydrogenase, and an H₂ hydrogenase; or (iii) at least one exogenousnucleic acid encodes an enzyme selected from a CO dehydrogenase, an H₂hydrogenase, and combinations thereof.

In a further embodiment, the invention provides a non-naturallyoccurring microbial organism comprising a microbial organism having a3-hydroxyisobutyric acid pathway comprising at least one exogenousnucleic acid encoding a 3-hydroxyisobutyric acid pathway enzymeexpressed in a sufficient amount to produce 3-hydroxyisobutyric acid;wherein said 2-hydroxyisobutyric acid pathway enzymes are selected fromthose shown in FIG. 16B; said non-naturally occurring microbial organismfurther comprising (i) a reductive TCA pathway comprising at least oneexogenous nucleic acid encoding a reductive TCA pathway enzyme, whereinsaid at least one exogenous nucleic acid is selected from an ATP-citratelyase, citrate lyase, a fumarate reductase, and analpha-ketoglutarate:ferredoxin oxidoreductase, or optionally isocitratedehydrogenase, aconitase, citryl-CoA synthetase or citryl-CoA lyase;(ii) a reductive TCA pathway comprising at least one exogenous nucleicacid encoding a reductive TCA pathway enzyme, wherein said at least oneexogenous nucleic acid is selected from a pyruvate:ferredoxinoxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvatecarboxykinase, a CO dehydrogenase, and an H₂ hydrogenase; or (iii) atleast one exogenous nucleic acid encodes an enzyme selected from a COdehydrogenase, an H₂ hydrogenase, and combinations thereof.

This invention is directed, in part, to improving the theoretical yieldsof products using syngas or its components, hydrogen, carbon dioxide andcarbon monoxide, as a source of reducing equivalents. In numerousengineered pathways, realization of maximum product yields based oncarbohydrate feedstock is hampered by insufficient reducing equivalentsor by loss of reducing equivalents and/or carbon to byproducts. Thetheoretical yields of several products on carbohydrate feedstocksincrease substantially if hydrogen and/or carbon monoxide can supplysufficient reducing equivalents. For example, the theoretical yields ofMAA, 2-hydroxyisobutyrate and 3-hydroxyisobutyrate increase to 2 mol/molglucose in the presence of H₂.

Reducing equivalents, or electrons, can be extracted from synthesis gascomponents such as CO and H₂ using carbon monoxide dehydrogenase (CODH)and hydrogenase enzymes, respectively. The reducing equivalents are thenpassed to acceptors such as oxidized ferredoxins, oxidized quinones,oxidized cytochromes, NADP+, water, or hydrogen peroxide to form reducedferredoxin, reduced quinones, reduced cytochromes, NAD(P)H, H₂, orwater, respectively. Reduced ferredoxin and NAD(P)H are particularlyuseful as they can serve as redox carriers for various Wood-Ljungdahlpathway and reductive TCA cycle enzymes.

Here, we show specific examples of how additional redox availabilityfrom CO and/or H₂ can improve the yields of reduced products such asmethacrylic acid, 2-hydroxyisobutyrate and 3-hydroxyisobutyrate.

Methacrylic acid (MAA), 3-hydroxybutyric acid and 2-hydroxyisobutyricacid are exemplary reduced products. The production of MAA throughfermentation has a theoretical yield of 1.33 moles MAA per mole ofglucose. It is most commonly produced from the acetone cyanohydrin (ACH)route using the raw materials acetone and hydrogen cyanide as rawmaterials. The intermediate cyanohydrin is converted with sulfuric acidto a sulfate ester of the methacrylamide, hydrolysis of which givesammonium bisulfate and MAA. Other producers start with an isobutyleneor, equivalently, tert-butanol, which is oxidized to methacrolein, andagain oxidized to methacrylic acid. MAA is then esterified with methanolto MMA. Methacrylic acid is used industrially in the preparation of itsesters, known collectively as methacrylates, such as methylmethacrylate. The methacrylates have numerous uses, most notably in themanufacture of polymers.C₆H₁₂O₆→1.33C₄H₆O₂+0.67CO₂+2H₂O

When the combined feedstocks strategy is applied to MAA production, thereducing equivalents generated from syngas can increase the MAAtheoretical yield from glucose to 2 mol MAA per mol of glucose with thepathways detailed in FIGS. 16A and 16B.1C₆H₁₂O₆+2CO₂+6H₂→2C₄H₆O₂+6H₂OOr1C₆H₁₂O₆+2CO+4H₂→2C₄H₆O₂+4H₂O

Similarly, the production of 3-hydroxyisobutyric acid throughfermentation can be improved by the combined feedstock strategy. Theproduction of 3-hydroxyisobutyric acid through fermentation has atheoretical yield of 1.33 mol 3-hydroxyisobutyric acid per mol ofglucose.3C₆H₁₂O₆→4C₄H₈O₃+2CO₂+2H₂O

When the combined feedstocks strategy is applied to 3-hydroxyisobutyricacid production, the reducing equivalents generated from syngas canincrease the 3-hydroxyisobutyric acid theoretical yield from glucose to2 mol 3-hydroxyisobutyric acid per mol of glucose with the pathwaysdetailed in FIGS. 16A and 16B.1C₆H₁₂O₆+2CO₂+6H₂→2C₄H₈O₃+4H₂O

Similarly, the production of 2-hydroxyisobutyric acid throughfermentation can be improved by the combined feedstock strategy. Theproduction of 2-hydroxyisobutyric acid through fermentation has atheoretical yield of 1.33 mol 2-hydroxyisobutyric acid per mol ofglucose.3C₆H₁₂O₆→4C₄H₈O₃+2CO₂+2H₂O

When the combined feedstocks strategy is applied to 3-hydroxyisobutyricacid production, the reducing equivalents generated from syngas canincrease the 3-hydroxyisobutyric acid theoretical yield from glucose to2 mol 2-hydroxyisobutyric acid per mol of glucose with the pathwaysdetailed in FIG. 16B.1C₆H₁₂O₆+2CO₂+6H₂→2C₄H₈O₃+4H₂O

The invention is also directed in part to increasing carbon flux throughthe central metabolism intermediate, acetyl-CoA, en route to productmolecules by enhancing carbon fixation via the reductive TCA cycle.Exemplary product molecules include methacrylic acid and2-hydroxyisobutyrate, although given the teachings and guidance providedherein, it will be recognized by one skilled in the art that any productmolecule that has acetyl-CoA as a building block can exhibit enhancedproduction through increased carbon flux through acetyl-CoA. The presentinvention provides non-naturally occurring microbial organisms havingone or more exogenous genes encoding enzymes that can catalyze variousenzymatic transformations en route to acetyl-CoA. In some embodiments,these enzymatic transformations are part of the reductive tricarboxylicacid (RTCA) cycle and are used to improve product yields fromcarbohydrate-based carbon feedstock. In other embodiments, theseenzymatic transformations are part of the Wood-Ljungdahl pathway.

The CO₂-fixing reductive tricarboxylic acid (RTCA) cycle is anendergenic anabolic pathway of CO₂ assimilation which uses NAD(P)H andATP. One turn of the RTCA cycle assimilates two moles of CO₂ into onemole of acetyl-CoA, or four moles of CO₂ into one mole of oxaloacetate.This additional availability of acetyl-CoA improves the maximumtheoretical yield of product molecules derived from carbohydrate-basedcarbon feedstock. Exemplary carbohydrates include but are not limited toglucose, sucrose, xylose, arabinose and glycerol. Note that the pathwaysfor the exemplary product molecules described herein all proceed throughacetyl-CoA.

The production of MAA from sugars via the pathways shown in FIGS. 16C,16D and 16E has a maximum yield of 1 mole MAA per mole glucose consumed,in the absence of reductive TCA cycle activity. The fixation of carbonby the reductive TCA cycle increases the yield of these pathways to themaximum theoretical yield of MAA from sugars. FIGS. 16C and 16D areexemplary flux distributions showing how additional carbon generated bythe reductive TCA cycle increases the yield of methacrylic acid producedby these pathways from 1 mole/mole glucose to 1.33 moles per moleglucose.C₆H₁₂O₆→1.33C₄H₆O₂+0.67CO₂+2H₂O

In one embodiment, the invention provides a non-naturally occurringmicrobial organism, comprising a microbial organism having amethacrylate ester pathway comprising at least one exogenous nucleicacid encoding a methacrylate ester pathway enzyme expressed in asufficient amount to produce a methacrylate ester; said non-naturallyoccurring microbial organism further comprising: (i) a reductive TCApathway comprising at least one exogenous nucleic acid encoding areductive TCA pathway enzyme, wherein said at least one exogenousnucleic acid is selected from an ATP-citrate lyase, a citrate lyase, afumarate reductase, and an alpha-ketoglutarate:ferredoxinoxidoreductase, or optionally isocitrate dehydrogenase, aconitase,citryl-CoA synthetase or citryl-CoA lyase; (ii) a reductive TCA pathwaycomprising at least one exogenous nucleic acid encoding a reductive TCApathway enzyme, wherein said at least one exogenous nucleic acid isselected from a pyruvate:ferredoxin oxidoreductase, aphosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, aCO dehydrogenase, and an H2 hydrogenase; or (iii) at least one exogenousnucleic acid encodes an enzyme selected from a CO dehydrogenase, an H2hydrogenase, and combinations thereof; wherein said methacrylate esterpathway comprises an alcohol transferase or an ester-forming enzyme, anda dehydratase. In one aspect, the dehydratase converts a3-hydroxyisobutyrate ester or a 2-hydroxyisobutyrate ester to saidmethacrylate ester. In one aspect, the alcohol transferase coverts3-hydroxyisobutyryl-CoA to a 3-hydroxyisobutyrate ester or2-hydroxyisobutyryl-CoA to 2-hydroxyisobutyrate ester.

In one embodiment, the invention provides a microbial organism, whereinthe microbial organism further comprises a methacyrlate ester pathwaycomprising at least one exogenous nucleic acid encoding a methacrylateester pathway enzyme expressed in a sufficient amount to produce amethacrylate ester, said methacrylate ester pathway comprising a pathwayselected from: (a) a 3-hydroxyisobutyrate-CoA transferase or a3-hydroxyisobutyrate-CoA synthetase; an alcohol transferase; and adehydratase; (b) a 3-hydroxyisobutyrate ester-forming enzyme and adehydratase; (c) a 2-hydroxyisobutyrate-CoA transferase or a2-hydroxyisobutyrate-CoA synthetase; an alcohol transferase; and adehydratase; or (d) a 2-hydroxyisobutyrate ester-forming enzyme and adehydratase. In another aspect, the microbial organism comprising (i)further comprises an exogenous nucleic acid encoding an enzyme selectedfrom a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitratedehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, afumarase, a malate dehydrogenase, an acetate kinase, aphosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxinoxidoreductase, ferredoxin, and combinations thereof. In another aspect,the microbial organism comprising (ii) further comprises an exogenousnucleic acid encoding an enzyme selected from an aconitase, anisocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoAtransferase, a fumarase, a malate dehydrogenase, and combinationsthereof.

In one aspect, the microbial organism comprises two exogenous nucleicacids each encoding a methacrylic acid pathway enzyme. In anotheraspect, the two exogenous nucleic acids encode an alcohol transferaseand a dehydratase or alternatively an ester-forming enzyme and adehydratase.

In one embodiment the invention provides a non-naturally occurringmicrobial, wherein said microbial organism comprising (i) comprises fourexogenous nucleic acids encoding an ATP-citrate lyase, citrate lyase, afumarate reductase, and an alpha-ketoglutarate:ferredoxinoxidoreductase; wherein said microbial organism comprising (ii)comprises five exogenous nucleic acids encoding a pyruvate:ferredoxinoxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvatecarboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or whereinsaid microbial organism comprising (iii) comprises two exogenous nucleicacids encoding a CO dehydrogenase and an H2 hydrogenase.

In one embodiment, the invention provide a method for producing amethacrylate ester comprising, culturing the non-naturally occurringmicrobial organism as described herein under conditions and for asufficient period of time to produce methacrylate ester.

The invention is described herein with general reference to themetabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing, or a protein associated with, thereferenced metabolic reaction, reactant or product. Unless otherwiseexpressly stated herein, those skilled in the art will understand thatreference to a reaction also constitutes reference to the reactants andproducts of the reaction. Similarly, unless otherwise expressly statedherein, reference to a reactant or product also references the reaction,and reference to any of these metabolic constituents also references thegene or genes encoding the enzymes that catalyze or proteins involved inthe referenced reaction, reactant or product. Likewise, given the wellknown fields of metabolic biochemistry, enzymology and genomics,reference herein to a gene or encoding nucleic acid also constitutes areference to the corresponding encoded enzyme and the reaction itcatalyzes or a protein associated with the reaction as well as thereactants and products of the reaction.

As disclosed herein, the product methacrylic acid, as well as otherintermediates, are carboxylic acids, which can occur in various ionizedforms, including fully protonated, partially protonated, and fullydeprotonated forms. Accordingly, the suffix “-ate,” or the acid form,can be used interchangeably to describe both the free acid form as wellas any deprotonated form, in particular since the ionized form is knownto depend on the pH in which the compound is found. It is understoodthat carboxylate products or intermediates includes ester forms ofcarboxylate products or pathway intermediates, such as O-carboxylate andS-carboxylate esters. O- and S-carboxylates can include lower alkyl,that is C1 to C6, branched or straight chain carboxylates. Some such O-or S-carboxylates include, without limitation, methyl, ethyl, n-propyl,n-butyl, i-propyl, sec-butyl, and tert-butyl, pentyl, hexyl O- orS-carboxylates, any of which can further possess an unsaturation,providing for example, propenyl, butenyl, pentyl, and hexenyl O- orS-carboxylates. O-carboxylates can be the product of a biosyntheticpathway. Exemplary O-carboxylates accessed via biosynthetic pathways caninclude, without limitation, methyl methacrylate, ethyl methacrylate,and n-propyl methacrylate. Other biosynthetically accessibleO-carboxylates can include medium to long chain groups, that is C7-C22,O-carboxylate esters derived from fatty alcohols, such heptyl, octyl,nonyl, decyl, undecyl, lauryl, tridecyl, myristyl, pentadecyl, cetyl,palmitolyl, heptadecyl, stearyl, nonadecyl, arachidyl, heneicosyl, andbehenyl alcohols, any one of which can be optionally branched and/orcontain unsaturations. O-carboxylate esters can also be accessed via achemical process, such as esterification of a free carboxylic acidproduct or transesterification of an O- or S-carboxylate. S-carboxylatesare exemplified by CoA S-esters, cysteinyl S-esters, alkylthioesters,and various aryl and heteroaryl thioesters. Additionally, the formationof methacrylic acid esters via a methacryl-CoA intermediate has beenproposed (WO/2007/039415 and U.S. Pat. No. 7,901,915). Methacryl-CoA canbe formed from methacrylate using a methacryl-CoA synthetase asdescribed in WO/2007/039415 and U.S. Pat. No. 7,901,915 or by applying amethacryl-CoA transferase (see FIG. 2 and Example III). Amethacrylyl-CoA transferase can transfer a CoA moiety to methacrylatefrom several CoA donors including, but not limited to, acetyl-CoA,succinyl-CoA, butyryl-CoA, and propionyl-CoA. Methacrylate can be formedfrom the pathways depicted in FIG. 1 or the pathways described inWO/2009/135074 or U.S. publication 2009/0275096. Methacrylyl-CoA canalternatively be produced from 2-hydroxyisobutyryl-CoA,3-hydroxyisobutyryl-CoA, 3-hydroxyisobutyrate, or isobutyryl-CoA via2-hydroxyisobutyryl-CoA dehydratase, 3-hydroxyisobutyryl-CoAdehydratase, 3-hydroxyisobutyrate dehydratase, or isobutyryl-CoAdehydrogenase as described in WO/2009/135074.

Thus, the invention additionally provides microbial organisms forproducing methacrylate esters. Such organisms can comprise amethacrylate ester pathway comprising methacrylyl-CoA transferase. Sucha microbial organism can further comprise a methacrylate ester pathwaycomprising methacrylyl-CoA synthetase and/or alcohol transferase (seeFIG. 2 and Example III). It is understood that such an organism thatproduces a methacrylate ester can be engineered to contain a microbialorganism containing a methacrylic acid or methacrylyl-CoA pathway,including but not limited to the methacrylic acid pathways disclosedherein (see Examples I and V-XIV and FIGS. 1-11) or described inWO2009/135074 or U.S. publication 2009/0275096. Thus, any of thedisclosed microbial organisms that produce methacrylic acid can furthercomprise at least one exogenous nucleic acid encoding a methacrylateester pathway enzyme expressed in a sufficient amount to producemethacrylate ester, the methacrylate ester pathway comprisingmethacrylyl-CoA synthetase, methacrylyl-CoA transferase and alcoholtransferase. Exemplary enzymatic and chemical conversion of methacrylicacid to methacrylate esters is described in Example IV.

Thus, the invention additionally provides a microbial organismcomprising a methacrylate ester pathway and further comprising amethacrylic acid pathway disclosed herein, including the methacrylicacid pathways described in FIGS. 1 and 3-11 and in Examples I and V-XIV.In a particular embodiment, the microbial organism comprising amethacrylate ester pathway further comprises a methacrylic acid pathwayselected from 3-hydroxyisobutyrate dehydratase; methylmalonyl-CoAmutase, methylmalonyl-CoA reductase, 3-hydroxyisobutyrate dehydrogenase,and 3-hydroxyisobutyrate dehydratase; methylmalonyl-CoA mutase,methylmalonyl-CoA epimerase, methylmalonyl-CoA reductase,3-hydroxyisobutyrate dehydrogenase, and 3-hydroxyisobutyratedehydratase; methylmalonyl-CoA mutase, alcohol/aldehyde dehydrogenase,and 3-hydroxyisobutyrate dehydratase; methylmalonyl-CoA mutase,methylmalonyl-CoA epimerase, alcohol/aldehyde dehydrogenase, and3-hydroxyisobutyrate dehydratase; methylmalonyl-CoA mutase,methylmalonyl-CoA reductase, 3-amino-2-methylpropionate transaminase,and 3-amino-2-methylpropionate ammonia lyase; methylmalonyl-CoA mutase,methylmalonyl-CoA epimerase, methylmalonyl-CoA reductase,3-amino-2-methylpropionate transaminase, and 3-amino-2-methylpropionateammonia lyase; 4-hydroxybutyryl-CoA mutase, 3-hydroxyisobutyryl-CoAsynthetase or 3-hydroxyisobutyryl-CoA hydrolase or3-hydroxyisobutyryl-CoA transferase, and 3-hydroxyisobutyratedehydratase; aspartate aminotransferase, glutamate mutase,3-methylaspartase, and mesaconate decarboxylase; alpha-ketoglutaratereductase, 2-hydroxyglutamate mutase, 3-methylmalate dehydratase, andmesaconate decarboxylase; acetoacetyl-CoA thiolase, acetoacetyl-CoAreductase, 3-hydroxybutyryl-CoA mutase, 2-hydroxyisobutyryl-CoAdehydratase, and methacrylyl-CoA transferase or methacrylyl-CoAhydrolase or methacrylyl-CoA synthetase; acetoacetyl-CoA thiolase,acetoacetyl-CoA reductase, 3-hydroxybutyryl-CoA mutase,2-hydroxyisobutyryl-CoA dehydratase, enoyl-CoA hydratase, and3-hydroxyisobutyryl-CoA hydrolase or 3-hydroxyisobutyryl-CoA synthetaseor 3-hydroxyisobutyryl-CoA transferase, and 3-hydroxyisobutyratedehydratase; 4-hydroxybutyryl-CoA dehydratase, vinylacetyl-CoAΔ-isomerase, crotonase, 3-hydroxybutyryl-CoA mutase,2-hydroxyisobutyryl-CoA dehydratase, and any of methacrylyl-CoAhydrolase or methacrylyl-CoA synthetase or methacrylyl-CoA transferase;and 4-hydroxybutyryl-CoA mutase, 3-hydroxyisobutyryl-CoA dehydratase,and methacrylyl-CoA synthetase or methacrylyl-CoA hydrolase ormethacrylyl-CoA transferase (see Examples V-XIV).

Exemplary non-naturally occurring microbial organisms capable ofproducing methacrylic acid are disclosed herein. For example, amethacrylic acid pathway is provided in which succinyl-CoA is aprecursor (see Examples V-VI, FIGS. 3 and 4). In one embodiment, anon-naturally occurring microbial organism has a methacrylic acidpathway comprising at least one exogenous nucleic acid encoding amethacrylic acid pathway enzyme expressed in a sufficient amount toproduce methacrylic acid, the methacrylic acid pathway comprisingmethylmalonyl-CoA mutase, methylmalonyl-CoA epimerase, methylmalonyl-CoAreductase, 3-hydroxyisobutyrate dehydrogenase and 3-hydroxyisobutyratedehydratase (see Examples V and VI and FIG. 3). In another embodiment, anon-naturally occurring microbial organism has a methacrylic acidpathway comprising at least one exogenous nucleic acid encoding amethacrylic acid pathway enzyme expressed in a sufficient amount toproduce methacrylic acid, the methacrylic acid pathway comprisingmethylmalonyl-CoA mutase, methylmalonyl-CoA epimerase, alcohol/aldehydedehydrogenase, and 3-hydroxyisobutyrate dehydratase (see Example V).Additionally, a non-naturally occurring microbial organism can have amethacrylic acid pathway comprising at least one exogenous nucleic acidencoding a methacrylic acid pathway enzyme expressed in a sufficientamount to produce methacrylic acid, the methacrylic acid pathwaycomprising methylmalonyl-CoA mutase, methylmalonyl-CoA epimerase,methylmalonyl-CoA reductase, 3-amino-2-methylpropionate transaminase,and 3-amino-2-methylpropionate ammonia lyase (see Examples VI and FIG.4).

Additionally provided is a non-naturally occurring microbial organismcontaining a methacrylic acid pathway having 4-hydroxybutyryl-CoA as aprecursor. One such embodiment is a non-naturally occurring microbialorganism having a methacrylic acid pathway comprising at least oneexogenous nucleic acid encoding a methacrylic acid pathway enzymeexpressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising 4-hydroxybutyryl-CoA mutase,3-hydroxyisobutyryl-CoA synthetase or 3-hydroxyisobutyryl-CoA hydrolaseor 3-hydroxyisobutyryl-CoA transferase, and 3-hydroxyisobutyratedehydratase (see Examples VII and FIG. 5). Alternatively, the pathwaycould include 4-hydroxybutyryl-CoA mutase, 3-hydroxyisobutyryl-CoAdehydratase; and methacrylyl-CoA synthetase or methacrylyl-CoA hydrolaseor methacrylyl-CoA transferase.

Further provided is a non-naturally occurring microbial organismcontaining a methacrylic acid pathway having alpha-ketoglutarate as aprecursor. One such embodiment is a non-naturally occurring microbialorganism having a methacrylic acid pathway comprising at least oneexogenous nucleic acid encoding a methacrylic acid pathway enzymeexpressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising aspartate aminotransferase,glutamate mutase, 3-methylaspartase, and mesaconate decarboxylase (seeExamples VIII and FIG. 6). In yet another embodiment, a non-naturallyoccurring microbial organism has a methacrylic acid pathway comprisingat least one exogenous nucleic acid encoding a methacrylic acid pathwayenzyme expressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising alpha-ketoglutarate reductase,2-hydroxyglutamate mutase, 3-methylmalate dehydratase, and mesaconatedecarboxylase (see Examples IX and FIG. 7).

In still another embodiment, a non-naturally occurring microbialorganism containing a methacrylic acid pathway has acetyl-CoA as aprecursor. For example, a non-naturally occurring microbial organism canhave a methacrylic acid pathway comprising at least one exogenousnucleic acid encoding a methacrylic acid pathway enzyme expressed in asufficient amount to produce methacrylic acid, the methacrylic acidpathway comprising acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase,3-hydroxybutyryl-CoA mutase, 2-hydroxyisobutyryl-CoA dehydratase, andmethacrylyl-CoA transferase or methacrylyl-CoA hydrolase ormethacrylyl-CoA synthetase (see Examples X and FIG. 8). In anotherembodiment, a non-naturally occurring microbial organism hays amethacrylic acid pathway comprising at least one exogenous nucleic acidencoding a methacrylic acid pathway enzyme expressed in a sufficientamount to produce methacrylic acid, the methacrylic acid pathwaycomprising acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase,3-hydroxybutyryl-CoA mutase, 2-hydroxyisobutyryl-CoA dehydratase,enoyl-CoA hydratase, and 3-hydroxyisobutyryl-CoA hydrolase or3-hydroxyisobutyryl-CoA synthetase or 3-hydroxyisobutyryl-CoAtransferase, and 3-hydroxyisobutyrate dehydratase (see Example X).

In further embodiments, non-naturally occurring microbial organisms cancontain a methacrylic acid having 4-hydroxybutyryl-CoA as a precursor.For example, a non-naturally occurring microbial organism has amethacrylic acid pathway comprising at least one exogenous nucleic acidencoding a methacrylic acid pathway enzyme expressed in a sufficientamount to produce methacrylic acid, the methacrylic acid pathwaycomprising 4-hydroxybutyryl-CoA dehydratase; vinylacetyl-CoAΔ-isomerase; crotonase; 3-hydroxybutyryl-CoA mutase;2-hydroxyisobutyryl-CoA dehydratase; and methacrylyl-CoA hydrolase ormethacrylyl-CoA synthetase or methacrylyl-CoA transferase (see ExampleXIV and FIG. 8).

In yet another embodiment, a non-naturally occurring microbial organismhas a methacrylic acid pathway comprising at least one exogenous nucleicacid encoding a methacrylic acid pathway enzyme expressed in asufficient amount to produce methacrylic acid, the methacrylic acidpathway comprising acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase,crotonase, butyryl-CoA dehydrogenase, isobutyryl-CoA mutase,isobutyryl-CoA dehydrogenase, and methacrylyl-CoA synthetase ormethacrylyl-CoA hydrolase or methacrylyl-CoA transferase (see Example XIand FIG. 9).

Further provided is a non-naturally occurring microbial organismcontaining a methacrylic acid pathway having pyruvate as a precursor.For example, a non-naturally occurring microbial organism can have amethacrylic acid pathway comprising at least one exogenous nucleic acidencoding a methacrylic acid pathway enzyme expressed in a sufficientamount to produce methacrylic acid, the methacrylic acid pathwaycomprising lactate dehydrogenase, lactate-CoA transferase, lactoyl-CoAdehydratase, acyl-CoA dehydrogenase, propionyl-CoA carboxylase,methylmalonyl-CoA reductase, 3-hydroxyisobutyrate dehydrogenase, and3-hydroxyisobutyrate dehydratase (see Example XII and FIG. 10).

Also provided is a non-naturally occurring microbial organism containinga methacrylic acid pathway having 2-ketoisovalerate as a precursor. Forexample, a non-naturally occurring microbial organism can have amethacrylic acid pathway comprising at least one exogenous nucleic acidencoding a methacrylic acid pathway enzyme expressed in a sufficientamount to produce methacrylic acid, the methacrylic acid pathwaycomprising valine aminotransferase, 2-ketoisovalerate dehydrogenase,isobutyryl-CoA dehydrogenase, and methacrylyl-CoA synthetase ormethacrylyl-CoA hydrolase or methacrylyl-CoA transferase (see ExampleXIII and FIG. 11). Such a methacrylic acid pathway can further containvaline aminotransferase, which converst valine to 2-ketoisovalerate(FIG. 11). In addition, such a methacrylic acid pathway can furthercontain enzymes that convert pyruvate to 2-ketoisovalerate (FIG. 11),such as acetolactate synthase, acetohydroxy acid isomeroreductase anddihydroxy-acid dehydratase (see Example XIII).

The invention also provides a non-naturally occurring microbial organismhaving a methacrylate ester pathway comprising at least one exogenousnucleic acid encoding a methacrylate ester pathway enzyme expressed in asufficient amount to produce a methacrylate ester, the methacrylateester pathway comprising a methacrylyl-CoA transferase and an alcoholtransferase. In a further embodiment, such a methacrylate ester pathwaycan further comprise methacrylyl-CoA synthetase. In a particularembodiment, the microbial organism having a methacrylate ester pathwaycan comprise two exogenous nucleic acids encoding methacrylyl-CoA andalcohol transferase. In a still further embodiment, the inventionprovides a microbial organism having a methacrylate ester pathway cancomprise three exogenous nucleic acids encoding methacrylyl-CoAsynthetase, methacrylyl-CoA transferase, and alcohol transferase.

The non-naturally occurring microbial organisms of the invention can beproduced by introducing expressible nucleic acids encoding one or moreof the enzymes or proteins participating in one or more methacrylic acidbiosynthetic pathways. Depending on the host microbial organism chosenfor biosynthesis, nucleic acids for some or all of a particularmethacrylic acid biosynthetic pathway can be expressed. For example, ifa chosen host is deficient in one or more enzymes or proteins for adesired biosynthetic pathway, then expressible nucleic acids for thedeficient enzyme(s) or protein(s) are introduced into the host forsubsequent exogenous expression. Alternatively, if the chosen hostexhibits endogenous expression of some pathway genes, but is deficientin others, then an encoding nucleic acid is needed for the deficientenzyme(s) or protein(s) to achieve methacrylic acid biosynthesis. Thus,a non-naturally occurring microbial organism of the invention can beproduced by introducing exogenous enzyme or protein activities to obtaina desired biosynthetic pathway or a desired biosynthetic pathway can beobtained by introducing one or more exogenous enzyme or proteinactivities that, together with one or more endogenous enzymes orproteins, produces a desired product such as methacrylic acid.

Host microbial organisms can be selected from, and the non-naturallyoccurring microbial organisms generated in, for example, bacteria,yeast, fungus or any of a variety of other microorganisms applicable tofermentation processes. Exemplary bacteria include species selected fromEscherichia coli, Klebsiella oxytoca, Anaerobiospirillumsucciniciproducens, Actinobacillus succinogenes, Mannheimiasucciniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacteriumglutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcuslactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridiumacetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.Exemplary yeasts or fungi include species selected from Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis,Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichiapastoris, Rhizopus arrhizus, Rhizobus oryzae, Yarrowia lipolytica, andthe like. E. coli is a particularly useful host organisms since it is awell characterized microbial organism suitable for genetic engineering.Other particularly useful host organisms include yeast such asSaccharomyces cerevisiae. It is understood that any suitable microbialhost organism can be used to introduce metabolic and/or geneticmodifications to produce a desired product.

Depending on the methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate biosynthetic pathwayconstituents of a selected host microbial organism, the non-naturallyoccurring microbial organisms of the invention will include at least oneexogenously expressed methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate pathway-encodingnucleic acid and up to all encoding nucleic acids for one or moremethacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate biosynthetic pathways. For example, methacrylicacid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate biosynthesis can be established in a host deficientin a pathway enzyme or protein through exogenous expression of thecorresponding encoding nucleic acid. In a host deficient in all enzymesor proteins of a methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate pathway, exogenousexpression of all enzyme or proteins in the pathway can be included,although it is understood that all enzymes or proteins of a pathway canbe expressed even if the host contains at least one of the pathwayenzymes or proteins. For example, exogenous expression of all enzymes orproteins in a pathway for production of methacrylic acid can beincluded, such as citramalate synthase (A), citramalate dehydratase(citraconate forming) (B), and citraconate decarboxylase (C) (FIG. 1,pathway (1) A/B/C); citramalate synthase (A), citramalate dehydratase(citraconate forming) (B), citraconate isomerase (G), and mesaconatedecarboxylase (H) (FIG. 1, pathway (2) A/B/G/H); citramalate synthase(A), citramalate dehydratase (mesaconate forming) (F) citraconateisomerase (G), and citraconate decarboxylase (C) (FIG. 1, pathway (3)A/F/G/C); citramalate synthase (A), citramalate dehydratase (mesaconateforming) (F), and mesaconate decarboxylase (H) (FIG. 1, pathway (4)A/F/H); citramalyl-CoA lyase (D), citramalyl-CoA transferase, synthetaseor hydrolase (E), citramalate dehydratase (citraconate forming) (B), andcitraconate decarboxylase (C) (FIG. 1, pathway (5) D/E/B/C);citramalyl-CoA lyase (D), citramalyl-CoA transferase, synthetase orhydrolase (E), citramalate dehydratase (citraconate forming) (B),citraconate isomerase (G), and mesaconate decarboxylase (H) (FIG. 1,pathway (6) D/E/B/G/H); citramalyl-CoA lyase (D), citramalyl-CoAtransferase, synthetase or hydrolase (E), citramalate dehydratase(mesaconate forming) (F), and mesaconate decarboxylase (H) (FIG. 1,pathway (7) D/E/F/H); citramalyl-CoA lyase (D), citramalyl-CoAtransferase, synthetase or hydrolase (E), citramalate dehydratase(mesaconate forming) (F), citraconate isomerase (G), and citraconatedecarboxylase (C) (FIG. 1, pathway (8) D/E/F/G/C); aconitatedecarboxylase (I), itaconate isomerase (J), and citraconatedecarboxylase (C) (FIG. 1, pathway (9) I/J/C); aconitate decarboxylase(I), itaconate isomerase (J), citraconate isomerase (G), and mesaconatedecarboxylase (H) (FIG. 1, pathway (10) I/J/G/H); aconitatedecarboxylase (I), itaconyl-CoA transferase, synthetase or hydrolase(L), citramalyl-CoA dehydratase (K), citramalyl-CoA transferase,synthetase or hydrolase (E), citramalate dehydratase (citraconateforming) (B), and citraconate decarboxylase (C) (FIG. 1, pathway (11)I/L/K/E/B/C); aconitate decarboxylase (I), itaconyl-CoA transferase,synthetase or hydrolase (L), citramalyl-CoA dehydratase (K),citramalyl-CoA transferase, synthetase or hydrolase (E), citramalatedehydratase (citraconate forming) (B), citraconate isomerase (G), andmesaconate decarboxylase (H) (FIG. 1, pathway (12) I/L/K/E/B/G/H);aconitate decarboxylase (I), itaconyl-CoA transferase, synthetase orhydrolase (L), citramalyl-CoA dehydratase (K), citramalyl-CoAtransferase, synthetase or hydrolase (E), citramalate dehydratase(mesaconate forming) (F), and mesaconate decarboxylase (H) (FIG. 1,pathway (13) I/L/K/E/F/H); and aconitate decarboxylase (I), itaconyl-CoAtransferase, synthetase or hydrolase (L), citramalyl-CoA dehydratase(K), citramalyl-CoA transferase, synthetase or hydrolase (E),citramalate dehydratase (mesaconate forming) (F), citraconate isomerase(G), and citraconate decarboxylase (C) (FIG. 1, pathway (14)I/L/K/E/F/G/C).

Given the teachings and guidance provided herein, those skilled in theart will understand that the number of encoding nucleic acids tointroduce in an expressible form will, at least, parallel themethacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate pathway deficiencies of the selected host microbialorganism. Therefore, a non-naturally occurring microbial organism of theinvention can have one, two, three, four, five, six, or seven, dependingon the pathway, including up to all nucleic acids encoding the enzymesor proteins constituting a methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate biosynthetic pathwaydisclosed herein. In some embodiments, the non-naturally occurringmicrobial organisms also can include other genetic modifications thatfacilitate or optimize methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate biosynthesis or thatconfer other useful functions onto the host microbial organism. One suchother functionality can include, for example, augmentation of thesynthesis of one or more of the methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate pathway precursors suchas acetyl-CoA, pyruvate or aconitate.

Generally, a host microbial organism is selected such that it producesthe precursor of a methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate pathway, either as anaturally produced molecule or as an engineered product that eitherprovides de novo production of a desired precursor or increasedproduction of a precursor naturally produced by the host microbialorganism. For example, aconitate, acetyl-CoA, and pyruvate are producednaturally in a host organism such as E. coli. A host organism can beengineered to increase production of a precursor, as disclosed herein.In addition, a microbial organism that has been engineered to produce adesired precursor can be used as a host organism and further engineeredto express enzymes or proteins of a methacrylic acid, methacrylateester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate pathway.

In some embodiments, a non-naturally occurring microbial organism of theinvention is generated from a host that contains the enzymaticcapability to synthesize methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate. In this specificembodiment it can be useful to increase the synthesis or accumulation ofa methacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate pathway product to, for example, drive methacrylicacid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate pathway reactions toward methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrateproduction. Increased synthesis or accumulation can be accomplished by,for example, overexpression of nucleic acids encoding one or more of themethacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate pathway enzymes or proteins described herein. Overexpression of the enzyme or enzymes and/or protein or proteins of themethacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate pathway can occur, for example, through exogenousexpression of the endogenous gene or genes, or through exogenousexpression of the heterologous gene or genes. Therefore, naturallyoccurring organisms can be readily generated to be non-naturallyoccurring microbial organisms of the invention, for example, producingmethacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate, through overexpression of one, two, three, four,five, six or seven, depending on the number of enzymes in the pathway,that is, up to all nucleic acids encoding methacrylic acid, methacrylateester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate biosyntheticpathway enzymes or proteins. In addition, a non-naturally occurringorganism can be generated by mutagenesis of an endogenous gene thatresults in an increase in activity of an enzyme in the methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyratebiosynthetic pathway.

In particularly useful embodiments, exogenous expression of the encodingnucleic acids is employed. Exogenous expression confers the ability tocustom tailor the expression and/or regulatory elements to the host andapplication to achieve a desired expression level that is controlled bythe user. However, endogenous expression also can be utilized in otherembodiments such as by removing a negative regulatory effector orinduction of the gene's promoter when linked to an inducible promoter orother regulatory element. Thus, an endogenous gene having a naturallyoccurring inducible promoter can be up-regulated by providing theappropriate inducing agent, or the regulatory region of an endogenousgene can be engineered to incorporate an inducible regulatory element,thereby allowing the regulation of increased expression of an endogenousgene at a desired time. Similarly, an inducible promoter can be includedas a regulatory element for an exogenous gene introduced into anon-naturally occurring microbial organism.

It is understood that, in methods of the invention, any of the one ormore exogenous nucleic acids can be introduced into a microbial organismto produce a non-naturally occurring microbial organism of theinvention. The nucleic acids can be introduced so as to confer, forexample, a methacrylic acid, methacrylate ester, 3-hydroxyisobutyrateand/or 2-hydroxyisobutyrate biosynthetic pathway onto the microbialorganism. Alternatively, encoding nucleic acids can be introduced toproduce an intermediate microbial organism having the biosyntheticcapability to catalyze some of the required reactions to confermethacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate biosynthetic capability. For example, anon-naturally occurring microbial organism having a methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyratebiosynthetic pathway can comprise at least two exogenous nucleic acidsencoding desired enzymes or proteins, such as the combination ofcitramalate synthase and citraconate decarboxylase; citraconateisomerase and mesaconate decarboxylase; or itaconyl-CoA transferase,synthetase or hydrolase and itaconate isomerase, and the like. Thus, itis understood that any combination of two or more enzymes of abiosynthetic pathway can be included in a non-naturally occurringmicrobial organism of the invention. Similarly, it is understood thatany combination of three or more enzymes of a biosynthetic pathway canbe included in a non-naturally occurring microbial organism of theinvention, for example, citramalate dehydratase (citraconate forming),citraconate isomerase and mesaconate decarboxylase; citramalyl-CoAlyase, citramalyl-CoA transferase, synthetase or hydrolase andcitramalate dehydratase; or aconitate decarboxylase, itaconate isomeraseand mesaconate decarboxylase, and so forth, as desired, so long as thecombination of enzymes and/or proteins of the desired biosyntheticpathway results in production of the corresponding desired product.Similarly, any combination of four, for example, citramalyl-CoA lyase,citramalyl-CoA transferase, synthetase or hydrolase, citramalatedehydratase (citraconate forming) and mesaconate decarboxylase;aconitate decarboxylase, itaconate isomerase, citraconate isomerase andmesaconate decarboxylase, and the like, or more enzymes of abiosynthetic pathway as disclosed herein can be included in anon-naturally occurring microbial organism of the invention, as desired,including up to all enzymes in a pathway, so long as the combination ofenzymes of the desired biosynthetic pathway results in production of thecorresponding desired product.

In addition to the biosynthesis of methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate as described herein,the non-naturally occurring microbial organisms and methods of theinvention also can be utilized in various combinations with each otherand with other microbial organisms and methods well known in the art toachieve product biosynthesis by other routes. For example, onealternative to produce methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate other than use of themethacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate producers is through addition of another microbialorganism capable of converting a methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate pathway intermediate tomethacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate. One such procedure includes, for example, thefermentation of a microbial organism that produces a methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyratepathway intermediate. The methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate pathway intermediatecan then be used as a substrate for a second microbial organism thatconverts the methacrylic acid, methacrylate ester, 3-hydroxyisobutyrateand/or 2-hydroxyisobutyrate pathway intermediate to methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate.The methacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate pathway intermediate can be added directly toanother culture of the second organism or the original culture of themethacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate pathway intermediate producers can be depleted ofthese microbial organisms by, for example, cell separation, and thensubsequent addition of the second organism to the fermentation broth canbe utilized to produce the final product without intermediatepurification steps.

In other embodiments, the non-naturally occurring microbial organismsand methods of the invention can be assembled in a wide variety ofsubpathways to achieve biosynthesis of, for example, methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate. Inthese embodiments, biosynthetic pathways for a desired product of theinvention can be segregated into different microbial organisms, and thedifferent microbial organisms can be co-cultured to produce the finalproduct. In such a biosynthetic scheme, the product of one microbialorganism is the substrate for a second microbial organism until thefinal product is synthesized. For example, the biosynthesis ofmethacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate can be accomplished by constructing a microbialorganism that contains biosynthetic pathways for conversion of onepathway intermediate to another pathway intermediate or the product.Alternatively, methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate also can bebiosynthetically produced from microbial organisms through co-culture orco-fermentation using two organisms in the same vessel, where the firstmicrobial organism produces a methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate intermediate and thesecond microbial organism converts the intermediate to methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate.

Given the teachings and guidance provided herein, those skilled in theart will understand that a wide variety of combinations and permutationsexist for the non-naturally occurring microbial organisms and methods ofthe invention together with other microbial organisms, with theco-culture of other non-naturally occurring microbial organisms havingsubpathways and with combinations of other chemical and/or biochemicalprocedures well known in the art to produce methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate.

Sources of encoding nucleic acids for a methacrylic acid, methacrylateester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate pathway enzymeor protein can include, for example, any species where the encoded geneproduct is capable of catalyzing the referenced reaction. Such speciesinclude both prokaryotic and eukaryotic organisms including, but notlimited to, bacteria, including archaea and eubacteria, and eukaryotes,including yeast, plant, insect, animal, and mammal, including human.Exemplary species for such sources include, for example, Escherichiaspecies, including Escherichia coli, Escherichia fergusonii,Methanocaldococcus jannaschii, Leptospira interrrogans, Geobactersulfurreducens, Chloroflexus aurantiacus, Roseiflexus sp. RS-1,Chloroflexus aggregans, Achromobacter xylosoxydans, Clostrdia species,including Clostridium kluyveri, Clostridium symbiosum, Clostridiumacetobutylicum, Clostridium saccharoperbutylacetonicum, Clostridiumljungdahlii, Trichomonas vaginalis G3, Trypanosoma brucei,Acidaminococcus fermentans, Fusobacterium species, includingFusobacterium nucleatum, Fusobacterium mortiferum, Corynebacteriumglutamicum, Rattus norvegicus, Homo sapiens, Saccharomyces species,including Saccharomyces cerevisiae, Apsergillus species, includingAspergillus terreus, Aspergillus oryzae, Aspergillus niger, Gibberellazeae, Pichia stipitis, Mycobacterium species, including Mycobacteriumsmegmatis, Mycobacterium avium, including subsp. pratuberculosis,Salinispora arenicola Pseudomonas species, including Pseudomonas sp.CF600, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonasaeruginosa, Ralstonia species, including Ralstonia eutropha, Ralstoniaeutropha JMP134, Ralstonia eutropha H16, Ralstonia pickettii,Lactobacillus plantarum, Klebsiella oxytoca, Bacillus species, includingBacillus subtilis, Bacillus pumilus, Bacillus megaterium, Pedicoccuspentosaceus, Chlorofexus species, including Chloroflexus aurantiacus,Chloroflexus aggregans, Rhodobacter sphaeroides, Methanocaldococcusjannaschii, Leptospira interrrogans, Candida maltosa, Salmonellaspecies, including Salmonella enterica serovar Typhimurium, Shewanellaspecies, including Shewanella oneidensis, Shewanella sp. MR-4,Alcaligenes faecalis, Geobacillus stearothermophilus, Serratiamarcescens, Vibrio cholerae, Eubacterium barkeri, Bacteroidescapillosus, Archaeoglobus fulgidus, Archaeoglobus fulgidus, Haloarculamarismortui, Pyrobaculum aerophilum str. IM2, Rhizobium species,including Rhizobium leguminosarum, as well as other exemplary speciesdisclosed herein or available as source organisms for correspondinggenes. However, with the complete genome sequence available for now morethan 550 species (with more than half of these available on publicdatabases such as the NCBI), including 395 microorganism genomes and avariety of yeast, fungi, plant, and mammalian genomes, theidentification of genes encoding the requisite methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyratebiosynthetic activity for one or more genes in related or distantspecies, including for example, homologues, orthologs, paralogs andnonorthologous gene displacements of known genes, and the interchange ofgenetic alterations between organisms is routine and well known in theart. Accordingly, the metabolic alterations allowing biosynthesis ofmethacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate described herein with reference to a particularorganism such as E. coli can be readily applied to other microorganisms,including prokaryotic and eukaryotic organisms alike. Given theteachings and guidance provided herein, those skilled in the art willknow that a metabolic alteration exemplified in one organism can beapplied equally to other organisms.

In some instances, such as when an alternative methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyratebiosynthetic pathway exists in an unrelated species, methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyratebiosynthesis can be conferred onto the host species by, for example,exogenous expression of a paralog or paralogs from the unrelated speciesthat catalyzes a similar, yet non-identical metabolic reaction toreplace the referenced reaction. Because certain differences amongmetabolic networks exist between different organisms, those skilled inthe art will understand that the actual gene usage between differentorganisms may differ. However, given the teachings and guidance providedherein, those skilled in the art also will understand that the teachingsand methods of the invention can be applied to all microbial organismsusing the cognate metabolic alterations to those exemplified herein toconstruct a microbial organism in a species of interest that willsynthesize methacrylic acid, methacrylate ester, 3-hydroxyisobutyrateand/or 2-hydroxyisobutyrate.

Methods for constructing and testing the expression levels of anon-naturally occurring methacrylic acid-, methacrylate ester-,3-hydroxyisobutyrate- and/or 2-hydroxyisobutyrate-producing host can beperformed, for example, by recombinant and detection methods well knownin the art. Such methods can be found described in, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., ColdSpring Harbor Laboratory, New York (2001); and Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1999).

Exogenous nucleic acid sequences involved in a pathway for production ofmethacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate can be introduced stably or transiently into a hostcell using techniques well known in the art including, but not limitedto, conjugation, electroporation, chemical transformation, transduction,transfection, and ultrasound transformation. For exogenous expression inE. coli or other prokaryotic cells, some nucleic acid sequences in thegenes or cDNAs of eukaryotic nucleic acids can encode targeting signalssuch as an N-terminal mitochondrial or other targeting signal, which canbe removed before transformation into prokaryotic host cells, ifdesired. For example, removal of a mitochondrial leader sequence led toincreased expression in E. coli (Hoffmeister et al., J. Biol. Chem.280:4329-4338 (2005)). For exogenous expression in yeast or othereukaryotic cells, genes can be expressed in the cytosol without theaddition of leader sequence, or can be targeted to mitochondrion orother organelles, or targeted for secretion, by the addition of asuitable targeting sequence such as a mitochondrial targeting orsecretion signal suitable for the host cells. Thus, it is understoodthat appropriate modifications to a nucleic acid sequence to remove orinclude a targeting sequence can be incorporated into an exogenousnucleic acid sequence to impart desirable properties. Furthermore, genescan be subjected to codon optimization with techniques well known in theart to achieve optimized expression of the proteins.

An expression vector or vectors can be constructed to include one ormore methacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate biosynthetic pathway encoding nucleic acids asexemplified herein operably linked to expression control sequencesfunctional in the host organism. Expression vectors applicable for usein the microbial host organisms of the invention include, for example,plasmids, phage vectors, viral vectors, episomes and artificialchromosomes, including vectors and selection sequences or markersoperable for stable integration into a host chromosome. Additionally,the expression vectors can include one or more selectable marker genesand appropriate expression control sequences. Selectable marker genesalso can be included that, for example, provide resistance toantibiotics or toxins, complement auxotrophic deficiencies, or supplycritical nutrients not in the culture media. Expression controlsequences can include constitutive and inducible promoters,transcription enhancers, transcription terminators, and the like whichare well known in the art. When two or more exogenous encoding nucleicacids are to be co-expressed, both nucleic acids can be inserted, forexample, into a single expression vector or in separate expressionvectors. For single vector expression, the encoding nucleic acids can beoperationally linked to one common expression control sequence or linkedto different expression control sequences, such as one induciblepromoter and one constitutive promoter. The transformation of exogenousnucleic acid sequences involved in a metabolic or synthetic pathway canbe confirmed using methods well known in the art. Such methods include,for example, nucleic acid analysis such as Northern blots or polymerasechain reaction (PCR) amplification of mRNA, or immunoblotting forexpression of gene products, or other suitable analytical methods totest the expression of an introduced nucleic acid sequence or itscorresponding gene product. It is understood by those skilled in the artthat the exogenous nucleic acid is expressed in a sufficient amount toproduce the desired product, and it is further understood thatexpression levels can be optimized to obtain sufficient expression usingmethods well known in the art and as disclosed herein.

The invention additionally provides methods of producing methacrylicacid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate using the microbial organisms of the inventioncomprising a methacrylic acid pathway. In a particular embodiment, theinvention provides a method for producing methacrylic acid by culturinga non-naturally occurring microbial organism, comprising a microbialorganism having a methacrylic acid pathway comprising at least oneexogenous nucleic acid encoding a methacrylic acid pathway enzymeexpressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising citramalate synthase (A),citramalate dehydratase (citraconate forming) (B), and citraconatedecarboxylase (C) (FIG. 1, pathway (1) A/B/C), under conditions and fora sufficient period of time to produce methacrylic acid. As disclosedherein, the microbial organism can comprise more than one exogenousnucleic acid encoding a methacrylic acid pathway enzyme, including up toall enzymes in a pathway, for example, three exogenous nucleic acidsencode citramalate synthase, citramalate dehydratase (citraconateforming), and citraconate decarboxylase.

In a particular embodiment, MAA producing non-naturally occurringmicrobial organism of the invention can have at least one exogenousnucleic acid that is a heterologous nucleic acid. In another embodimentthe non-naturally occurring microbial organism producing MAA can be in asubstantially anaerobic culture medium.

In another embodiment, the invention provides a method for producingmethacrylic acid by culturing a non-naturally occurring microbialorganism comprising a methacrylic acid pathway comprising at least oneexogenous nucleic acid encoding a methacrylic acid pathway enzymeexpressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising citramalate synthase (A),citramalate dehydratase (citraconate forming) (B), citraconate isomerase(G), and mesaconate decarboxylase (H) (FIG. 1, pathway (2) A/B/G/H),under conditions and for a sufficient period of time to producemethacrylic acid. In still another embodiment, the invention provides amethod for producing methacrylic acid by culturing a non-naturallyoccurring microbial organism, comprising a methacrylic acid pathwaycomprising at least one exogenous nucleic acid encoding a methacrylicacid pathway enzyme expressed in a sufficient amount to producemethacrylic acid, the methacrylic acid pathway comprising citramalatesynthase (A), citramalate dehydratase (mesaconate forming) (F)citraconate isomerase (G), and citraconate decarboxylase (C) (FIG. 1,pathway (3) A/F/G/C), under conditions and for a sufficient period oftime to produce methacrylic acid.

In a further embodiment, the invention provides a method for producingmethacrylic acid by culturing a non-naturally occurring microbialorganism, comprising a methacrylic acid pathway comprising at least oneexogenous nucleic acid encoding a methacrylic acid pathway enzymeexpressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising citramalate synthase (A),citramalate dehydratase (mesaconate forming) (F), and mesaconatedecarboxylase (H) (FIG. 1, pathway (4) A/F/H), under conditions and fora sufficient period of time to produce methacrylic acid. In yet afurther embodiment, the invention provides a method for producingmethacylic acid by culturing a non-naturally occurring microbialorganism, comprising a methacrylic acid pathway comprising at least oneexogenous nucleic acid encoding a methacrylic acid pathway enzymeexpressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising citramalyl-CoA lyase (D),citramalyl-CoA transferase, synthetase or hydrolase (E), citramalatedehydratase (citraconate forming) (B), and citraconate decarboxylase (C)(FIG. 1, pathway (5) D/E/B/C), under conditions and for a sufficientperiod of time to produce methacrylic acid.

In an additional embodiment, the invention provides a method forproducing methacrylic acid by culturing a non-naturally occurringmicrobial organism, comprising a methacrylic acid pathway comprising atleast one exogenous nucleic acid encoding a methacrylic acid pathwayenzyme expressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising citramalyl-CoA lyase (D),citramalyl-CoA transferase, synthetase or hydrolase (E), citramalatedehydratase (citraconate forming) (B), citraconate isomerase (G), andmesaconate decarboxylase (H) (FIG. 1, pathway (6) D/E/B/G/H), underconditions and for a sufficient period of time to produce methacrylicacid. In still another embodiment, the invention provides a method forproducing methacrylic acid by culturing a non-naturally occurringmicrobial organism, comprising a methacrylic acid pathway comprising atleast one exogenous nucleic acid encoding a methacrylic acid pathwayenzyme expressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising citramalyl-CoA lyase (D),citramalyl-CoA transferase, synthetase or hydrolase (E), citramalatedehydratase (mesaconate forming) (F), and mesaconate decarboxylase (H)(FIG. 1, pathway (7) D/E/F/H), under conditions and for a sufficientperiod of time to produce methacrylic acid.

In another embodiment, the invention provides a method for producingmethacrylic acid by culturing a non-naturally occurring microbialorganism, comprising a methacrylic acid pathway comprising at least oneexogenous nucleic acid encoding a methacrylic acid pathway enzymeexpressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising citramalyl-CoA lyase (D),citramalyl-CoA transferase, synthetase or hydrolase (E), citramalatedehydratase (mesaconate forming) (F), citraconate isomerase (G), andcitraconate decarboxylase (C) (FIG. 1, pathway (8) D/E/F/G/C), underconditions and for a sufficient period of time to produce methacrylicacid. Additionally, the invention provides a method for producingmethacrylic acid by culturing a non-naturally occurring microbialorganism, comprising a methacrylic acid pathway comprising at least oneexogenous nucleic acid encoding a methacrylic acid pathway enzymeexpressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising aconitate decarboxylase (I),itaconate isomerase (J), and citraconate decarboxylase (C) (FIG. 1,pathway (9) I/J/C), under conditions and for a sufficient period of timeto produce methacrylic acid.

In yet a further embodiment, the invention provides a method forproducing methacrylic acid by culturing a non-naturally occurringmicrobial organism, comprising a methacrylic acid pathway comprising atleast one exogenous nucleic acid encoding a methacrylic acid pathwayenzyme expressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising aconitate decarboxylase (I),itaconate isomerase (J), citraconate isomerase (G), and mesaconatedecarboxylase (H) (FIG. 1, pathway (10) I/J/G/H), under conditions andfor a sufficient period of time to produce methacrylic acid. In anadditional embodiment, the invention provides a method for producingmethacrylic acid by culturing a non-naturally occurring microbialorganism, comprising a methacrylic acid pathway comprising at least oneexogenous nucleic acid encoding a methacrylic acid pathway enzymeexpressed in a sufficient amount to produce methacrylic acid, themethacrylic acid pathway comprising aconitate decarboxylase (I),itaconyl-CoA transferase, synthetase or hydrolase (L), citramalyl-CoAdehydratase (K), citramalyl-CoA transferase, synthetase or hydrolase(E), citramalate dehydratase (citraconate forming) (B), and citraconatedecarboxylase (C) (FIG. 1, pathway (11) I/L/K/E/B/C), under conditionsand for a sufficient period of time to produce methacrylic acid.

The invention also provides a method for producing methacrylic acid byculturing a non-naturally occurring microbial organism, comprising amethacrylic acid pathway comprising at least one exogenous nucleic acidencoding a methacrylic acid pathway enzyme expressed in a sufficientamount to produce methacrylic acid, the methacrylic acid pathwaycomprising aconitate decarboxylase (I), itaconyl-CoA transferase,synthetase or hydrolase (L), citramalyl-CoA dehydratase (K),citramalyl-CoA transferase, synthetase or hydrolase (E), citramalatedehydratase (citraconate forming) (B), citraconate isomerase (G), andmesaconate decarboxylase (H) (FIG. 1, pathway (12) I/L/K/E/B/G/H), underconditions and for a sufficient period of time to produce methacrylicacid. In still a further embodiment, the invention provides a method forproducing methacrylic acid by culturing a non-naturally occurringmicrobial organism, comprising a microbial organism having a methacrylicacid pathway comprising at least one exogenous nucleic acid encoding amethacrylic acid pathway enzyme expressed in a sufficient amount toproduce methacrylic acid, the methacrylic acid pathway comprisingaconitate decarboxylase (I), itaconyl-CoA transferase, synthetase orhydrolase (L), citramalyl-CoA dehydratase (K), citramalyl-CoAtransferase, synthetase or hydrolase (E), citramalate dehydratase(mesaconate forming) (F), and mesaconate decarboxylase (H) (FIG. 1,pathway (13) I/L/K/E/F/H), under conditions and for a sufficient periodof time to produce methacrylic acid. In an additional embodiment, theinvention provides a method for producing methacrylic acid by culturinga non-naturally occurring microbial organism, comprising a methacrylicacid pathway comprising at least one exogenous nucleic acid encoding amethacrylic acid pathway enzyme expressed in a sufficient amount toproduce methacrylic acid, the methacrylic acid pathway comprisingaconitate decarboxylase (I), itaconyl-CoA transferase, synthetase orhydrolase (L), citramalyl-CoA dehydratase (K), citramalyl-CoAtransferase, synthetase or hydrolase (E), citramalate dehydratase(mesaconate forming) (F), citraconate isomerase (G), and citraconatedecarboxylase (C) (FIG. 1, pathway (14) I/L/K/E/F/G/C), under conditionsand for a sufficient period of time to produce methacrylic acid.

The invention also provides a method for producing a methacrylate esterby culturing a non-naturally occurring microbial organism underconditions and for a sufficient period of time to produce a3-hydroxyisobutyrate ester, wherein the non-naturally occurringmicrobial organism includes an exogenous nucleic acid encoding analcohol transferase or a 3-hydroxyisobutyrate ester-forming enzymeexpressed in a sufficient amount to produce a 3-hydroxyisobutyrateester, and chemically dehydrating the 3-hydroxyisobutyrate ester toproduce a methacrylate ester as exemplified in FIG. 28 and FIG. 30,steps 6 and 7. In another embodiment, the invention provides a methodfor producing methyl methacrylate by culturing a non-naturally occurringmicrobial organism under conditions and for a sufficient period of timeto produce methyl-3-hydroxyisobutyrate, wherein the non-naturallyoccurring microbial organism includes an exogenous nucleic acid encodingan alcohol transferase or an ester-forming enzyme expressed in asufficient amount to produce methyl-3-hydroxyisobutyrate, and chemicallydehydrating said methyl-3-hydroxyisobutyrate to produce methylmethacrylate as exemplified in FIG. 30, steps 6 and 7. In some aspectsof the invention, the methods for producing a methacrylate ester ormethyl methacrylate as disclosed herein further include that thenon-naturally occurring microbial organism includes at least oneexogenous nucleic acid encoding 3-hydroxyisobutyryl-CoA pathway enzymesas described in FIGS. 1-11 and 28-30 or any combination thereof. In someaspects, the invention provides that the exogenous nucleic acid is aheterologous nucleic acid. In some aspects, the invention provides thatthe non-naturally occurring microbial organism is in a substantiallyanaerobic culture medium.

In one embodiment, the invention provides a non-naturally occurringmicrobial organism, comprising a microbial organism having a methylmethacrylate pathway comprising at least one exogenous nucleic acidencoding a methyl methacrylate pathway enzyme expressed in a sufficientamount to produce methyl methacrylate; said non-naturally occurringmicrobial organism further comprising: (i) a reductive TCA pathwaycomprising at least one exogenous nucleic acid encoding a reductive TCApathway enzyme, wherein said at least one exogenous nucleic acid isselected from an ATP-citrate lyase, a citrate lyase, a fumaratereductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase, oroptionally isocitrate dehydrogenase, aconitase, citryl-CoA synthetase orcitryl-CoA lyase; (ii) a reductive TCA pathway comprising at least oneexogenous nucleic acid encoding a reductive TCA pathway enzyme, whereinsaid at least one exogenous nucleic acid is selected from apyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, aphosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H₂hydrogenase; or (iii) at least one exogenous nucleic acid encodes anenzyme selected from a CO dehydrogenase, an H₂ hydrogenase, andcombinations thereof; wherein said methyl methacrylate pathway comprisesan alcohol transferase or an ester-forming enzyme, and a dehydratase. Inanother aspect, the invention provides a method wherein the microbialorganism comprising (i) further comprises an exogenous nucleic acidencoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase,an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, asuccinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetatekinase, a phosphotransacetylase, an acetyl-CoA synthetase, anNAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof.In another aspect, the invention provides a method wherein the microbialorganism comprising (ii) further comprises an exogenous nucleic acidencoding an enzyme selected from an aconitase, an isocitratedehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, afumarase, a malate dehydrogenase, and combinations thereof. In oneaspect, the microbial organism comprises two exogenous nucleic acidseach encoding a methyl methacrylate enzyme. In a further aspect, the twoexogenous nucleic acids encode an alcohol transferase and a dehydrataseor alternatively a ester-forming enzyme and a dehydratase.

In one embodiment, the invention provides a method, wherein themicrobial organism comprising (i) comprises four exogenous nucleic acidsencoding an ATP-citrate lyase, citrate lyase, a fumarate reductase, andan alpha-ketoglutarate:ferredoxin oxidoreductase; wherein said microbialorganism comprising (ii) comprises five exogenous nucleic acids encodinga pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase,a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H₂hydrogenase; or wherein said microbial organism comprising (iii)comprises two exogenous nucleic acids encoding a CO dehydrogenase and anH₂ hydrogenase.

In one embodiment, the invention provides a method for producing methylmethacrylate comprising, culturing the non-naturally occurring microbialorganism as disclosed herein under conditions and for a sufficientperiod of time to produce methyl methacrylate.

In another embodiment, the invention provides a method for producing amethacrylate ester comprising, culturing a non-naturally occurringmicrobial organism under conditions and for a sufficient period of timeto produce a 3-hydroxyisobutyrate ester, wherein said non-naturallyoccurring microbial organism comprises an exogenous nucleic acidencoding an alcohol transferase or a 3-hydroxyisobutyrate ester-formingenzyme expressed in a sufficient amount to produce a3-hydroxyisobutyrate ester and said non-naturally occurring microbialorganism further comprising: (i) a reductive TCA pathway comprising atleast one exogenous nucleic acid encoding a reductive TCA pathwayenzyme, wherein said at least one exogenous nucleic acid is selectedfrom an ATP-citrate lyase, a citrate lyase, a fumarate reductase, and analpha-ketoglutarate:ferredoxin oxidoreductase, or optionally isocitratedehydrogenase, aconitase, citryl-CoA synthetase or citryl-CoA lyase;(ii) a reductive TCA pathway comprising at least one exogenous nucleicacid encoding a reductive TCA pathway enzyme, wherein said at least oneexogenous nucleic acid is selected from a pyruvate:ferredoxinoxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvatecarboxykinase, a CO dehydrogenase, and an H₂ hydrogenase; or (iii) atleast one exogenous nucleic acid encoding an enzyme selected from a COdehydrogenase, an H₂ hydrogenase, and combinations thereof, andchemically dehydrating said 3-hydroxyisobutyrate ester to produce amethacrylate ester. In another embodiment, the invention provides amethod for producing a methacrylate ester comprising, culturing anon-naturally occurring microbial organism under conditions and for asufficient period of time to produce a 2-hydroxyisobutyrate ester,wherein said non-naturally occurring microbial organism comprises anexogenous nucleic acid encoding an alcohol transferase or a2-hydroxyisobutyrate ester-forming enzyme expressed in a sufficientamount to produce a 2-hydroxy isobutyrate ester and said non-naturallyoccurring microbial organism further comprising: (i) a reductive TCApathway comprising at least one exogenous nucleic acid encoding areductive TCA pathway enzyme, wherein said at least one exogenousnucleic acid is selected from an ATP-citrate lyase, a citrate lyase, afumarate reductase, and an alpha-ketoglutarate:ferredoxinoxidoreductase, or optionally isocitrate dehydrogenase, aconitase,citryl-CoA synthetase or citryl-CoA lyase; (ii) a reductive TCA pathwaycomprising at least one exogenous nucleic acid encoding a reductive TCApathway enzyme, wherein said at least one exogenous nucleic acid isselected from a pyruvate:ferredoxin oxidoreductase, aphosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, aCO dehydrogenase, and an H₂ hydrogenase; or (iii) at least one exogenousnucleic acid encoding an enzyme selected from a CO dehydrogenase, an H₂hydrogenase, and combinations thereof, and chemically dehydrating said2-hydroxy isobutyrate ester to produce a methacrylate ester. In yetanother embodiment, the invention provides a method for producing methylmethacrylate comprising, culturing a non-naturally occurring microbialorganism under conditions and for a sufficient period of time to producemethyl-3-hydroxyisobutyrate, wherein said non-naturally occurringmicrobial organism comprises an exogenous nucleic acid encoding analcohol transferase or an ester-forming enzyme expressed in a sufficientamount to produce methyl-3-hydroxyisobutyrate and said non-naturallyoccurring microbial organism further comprising: (i) a reductive TCApathway comprising at least one exogenous nucleic acid encoding areductive TCA pathway enzyme, wherein said at least one exogenousnucleic acid is selected from an ATP-citrate lyase, a citrate lyase, afumarate reductase, and an alpha-ketoglutarate:ferredoxinoxidoreductase, or optionally isocitrate dehydrogenase, aconitase,citryl-CoA synthetase or citryl-CoA lyase; (ii) a reductive TCA pathwaycomprising at least one exogenous nucleic acid encoding a reductive TCApathway enzyme, wherein said at least one exogenous nucleic acid isselected from a pyruvate:ferredoxin oxidoreductase, aphosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, aCO dehydrogenase, and an H₂ hydrogenase; or (iii) at least one exogenousnucleic acid encoding an enzyme selected from a CO dehydrogenase, an H₂hydrogenase, and combinations thereof, and chemically dehydrating saidmethyl-3-hydroxyisobutyrate to produce methyl methacrylate. In oneaspect of the above methods, at least one of said exogenous nucleicacids is a heterologous nucleic acid. In another aspect of the abovemethods, the non-naturally occurring microbial organism is in asubstantially anaerobic culture medium.

In one embodiment, the invention provides a method as disclosed aboveand herein, wherein said microbial organism comprising (i) furthercomprises an exogenous nucleic acid encoding an enzyme selected from apyruvate:ferredoxin oxidoreductase, an aconitase, an isocitratedehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, afumarase, a malate dehydrogenase, an acetate kinase, aphosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxinoxidoreductase, ferredoxin, and combinations thereof. In anotherembodiment, the invention provides a method as disclosed above andherein, wherein said microbial organism comprising (ii) furthercomprises an exogenous nucleic acid encoding an enzyme selected from anaconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, asuccinyl-CoA transferase, a fumarase, a malate dehydrogenase, andcombinations thereof. In yet another embodiment, the invention providesa method as disclosed above and herein, wherein said microbial organismcomprising (i) comprises four exogenous nucleic acids encoding anATP-citrate lyase, citrate lyase, a fumarate reductase, and analpha-ketoglutarate:ferredoxin oxidoreductase; wherein said microbialorganism comprising (ii) comprises five exogenous nucleic acids encodinga pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase,a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H₂hydrogenase; or wherein said microbial organism comprising (iii)comprises two exogenous nucleic acids encoding a CO dehydrogenase and anH₂ hydrogenase.

In one aspect, the methods disclosed herein can further a microbialorganism comprising a 3-hydroxyisobutyrate ester pathway comprising atleast one exogenous nucleic acid encoding a 3-hydroxyisobutyrate esterpathway enzyme expressed in a sufficient amount to produce a3-hydroxyisobutyrate ester, said 3-hydroxyisobutyrate ester pathwaycomprising a pathway selected from: (a) a 3-hydroxyisobutyrate-CoAtransferase or a 3-hydroxyisobutyrate-CoA synthetase; and an alcoholtransferase; or (b) 3-hydroxyisobutyrate ester-forming enzyme. In oneaspect, the methods disclosed herein can further a microbial organismcomprising a 2-hydroxyisobutyrate ester pathway comprising at least oneexogenous nucleic acid encoding a 2-hydroxyisobutyrate ester pathwayenzyme expressed in a sufficient amount to produce a2-hydroxyisobutyrate ester, said 2-hydroxyisobutyrate ester pathwaycomprising a pathway selected from: (a) a 2-hydroxyisobutyrate-CoAtransferase or a 2-hydroxyisobutyrate-CoA synthetase; and an alcoholtransferase; or (b) a 2-hydroxyisobutyrate ester-forming enzyme.

Suitable purification and/or assays to test for the production ofmethacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate can be performed using well known methods. Suitablereplicates such as triplicate cultures can be grown for each engineeredstrain to be tested. For example, product and byproduct formation in theengineered production host can be monitored. The final product andintermediates, and other organic compounds, can be analyzed by methodssuch as HPLC (High Performance Liquid Chromatography), GC-MS (GasChromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-MassSpectroscopy) or other suitable analytical methods using routineprocedures well known in the art. The release of product in thefermentation broth can also be tested with the culture supernatant.Byproducts and residual glucose can be quantified by HPLC using, forexample, a refractive index detector for glucose and alcohols, and a UVdetector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779(2005)), or other suitable assay and detection methods well known in theart. The individual enzyme or protein activities from the exogenous DNAsequences can also be assayed using methods well known in the art. Forexample, citramalate synthase activity can be assayed by monitoring theproduction of CoA over time in a solution of purified protein,acetyl-CoA, pyruvate and buffer (Atsumi et al., AEM74:7802-7808 (2008)).

The methacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate can be separated from other components in theculture using a variety of methods well known in the art. Suchseparation methods include, for example, extraction procedures as wellas methods that include continuous liquid-liquid extraction,pervaporation, membrane filtration, membrane separation, reverseosmosis, electrodialysis, distillation, crystallization, centrifugation,extractive filtration, ion exchange chromatography, size exclusionchromatography, adsorption chromatography, and ultrafiltration. All ofthe above methods are well known in the art.

Any of the non-naturally occurring microbial organisms described hereincan be cultured to produce and/or secrete the biosynthetic products ofthe invention. For example, the methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate producers can becultured for the biosynthetic production of methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate.

For the production of methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate, the recombinantstrains are cultured in a medium with carbon source and other essentialnutrients. It is sometimes desirable and can be highly desirable tomaintain anaerobic conditions in the fermenter to reduce the cost of theoverall process. Such conditions can be obtained, for example, by firstsparging the medium with nitrogen and then sealing the flasks with aseptum and crimp-cap. For strains where growth is not observedanaerobically, microaerobic or substantially anaerobic conditions can beapplied by perforating the septum with a small hole for limitedaeration. Exemplary anaerobic conditions have been described previouslyand are well-known in the art. Exemplary aerobic and anaerobicconditions are described, for example, in United State publication2009/0047719, filed Aug. 10, 2007. Fermentations can be performed in abatch, fed-batch or continuous manner, as disclosed herein.

If desired, the pH of the medium can be maintained at a desired pH, inparticular neutral pH, such as a pH of around 7 by addition of a base,such as NaOH or other bases, or acid, as needed to maintain the culturemedium at a desirable pH. The growth rate can be determined by measuringoptical density using a spectrophotometer (600 nm), and the glucoseuptake rate by monitoring carbon source depletion over time.

The growth medium can include, for example, any carbohydrate sourcewhich can supply a source of carbon to the non-naturally occurringmicroorganism. Such sources include, for example, sugars such asglucose, xylose, arabinose, galactose, mannose, fructose, sucrose andstarch. Other sources of carbohydrate include, for example, renewablefeedstocks and biomass. Exemplary types of biomasses that can be used asfeedstocks in the methods of the invention include cellulosic biomass,hemicellulosic biomass and lignin feedstocks or portions of feedstocks.Such biomass feedstocks contain, for example, carbohydrate substratesuseful as carbon sources such as glucose, xylose, arabinose, galactose,mannose, fructose and starch. Given the teachings and guidance providedherein, those skilled in the art will understand that renewablefeedstocks and biomass other than those exemplified above also can beused for culturing the microbial organisms of the invention for theproduction of methacrylic acid, methacrylate ester, 3-hydroxyisobutyrateand/or 2-hydroxyisobutyrate.

In some embodiments, the present invention provides methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate ora methacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate intermediate that has a carbon-12, carbon-13, andcarbon-14 ratio that reflects an atmospheric carbon uptake source. Insome such embodiments, the uptake source is CO₂. In some embodiments, Insome embodiments, the present invention provides methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate ora methacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate intermediate that has a carbon-12, carbon-13, andcarbon-14 ratio that reflects petroleum-based carbon uptake source. Insome embodiments, the present invention provides methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate ora methacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate intermediate that has a carbon-12, carbon-13, andcarbon-14 ratio that is obtained by a combination of an atmosphericcarbon uptake source with a petroleum-based uptake source. Suchcombination of uptake sources is one means by which the carbon-12,carbon-13, and carbon-14 ratio can be varied.

In addition to renewable feedstocks such as those exemplified above andherein, the methacrylic acid methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate microbial organisms ofthe invention also can be modified for growth on syngas as its source ofcarbon. In this specific embodiment, one or more proteins or enzymes areexpressed in the methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate producing organisms toprovide a metabolic pathway for utilization of syngas or other gaseouscarbon source.

Synthesis gas, also known as syngas or producer gas, is the majorproduct of gasification of coal and of carbonaceous materials such asbiomass materials, including agricultural crops and residues. Syngas isa mixture primarily of H₂ and CO and can be obtained from thegasification of any organic feedstock, including but not limited tocoal, coal oil, natural gas, biomass, and waste organic matter.Gasification is generally carried out under a high fuel to oxygen ratio.Although largely H₂ and CO, syngas can also include CO₂ and other gasesin smaller quantities. Thus, synthesis gas provides a cost effectivesource of gaseous carbon such as CO and, additionally, CO₂.

The Wood-Ljungdahl pathway catalyzes the conversion of CO and H₂ toacetyl-CoA and other products such as acetate. Organisms capable ofutilizing CO and syngas also generally have the capability of utilizingCO₂ and CO₂/H₂ mixtures through the same basic set of enzymes andtransformations encompassed by the Wood-Ljungdahl pathway. H₂-dependentconversion of CO₂ to acetate by microorganisms was recognized longbefore it was revealed that CO also could be used by the same organismsand that the same pathways were involved. Many acetogens have been shownto grow in the presence of CO₂ and produce compounds such as acetate aslong as hydrogen is present to supply the necessary reducing equivalents(see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, NewYork, (1994)). This can be summarized by the following equation:2CO₂+4H₂ +nADP+nPi→CH₃COOH+2H₂O+nATPHence, non-naturally occurring microorganisms possessing theWood-Ljungdahl pathway can utilize CO₂ and H₂ mixtures as well for theproduction of acetyl-CoA and other desired products.

The Wood-Ljungdahl pathway is well known in the art and consists of 12reactions which can be separated into two branches: (1) methyl branchand (2) carbonyl branch. The methyl branch converts syngas tomethyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branchconverts methyl-THF to acetyl-CoA. The reactions in the methyl branchare catalyzed in order by the following enzymes or proteins: ferredoxinoxidoreductase, formate dehydrogenase, formyltetrahydrofolatesynthetase, methenyltetrahydrofolate cyclodehydratase,methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolatereductase. The reactions in the carbonyl branch are catalyzed in orderby the following enzymes or proteins: methyltetrahydrofolate:corrinoidprotein methyltransferase (for example, AcsE), corrinoid iron-sulfurprotein, nickel-protein assembly protein (for example, AcsF),ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase andnickel-protein assembly protein (for example, CooC). Following theteachings and guidance provided herein for introducing a sufficientnumber of encoding nucleic acids to generate a methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyratepathway, those skilled in the art will understand that the sameengineering design also can be performed with respect to introducing atleast the nucleic acids encoding the Wood-Ljungdahl enzymes or proteinsabsent in the host organism. Therefore, introduction of one or moreencoding nucleic acids into the microbial organisms of the inventionsuch that the modified organism contains the complete Wood-Ljungdahlpathway will confer syngas utilization ability.

Additionally, the reductive (reverse) tricarboxylic acid cycle is and/orhydrogenase activities can also be used for the conversion of CO, CO₂and/or H₂ to acetyl-CoA and other products such as acetate. Organismscapable of fixing carbon via the reductive TCA pathway can utilize oneor more of the following enzymes: ATP citrate-lyase, citrate lyase,aconitase, isocitrate dehydrogenase, alpha-ketoglutarate:ferredoxinoxidoreductase, succinyl-CoA synthetase, succinyl-CoA transferase,fumarate reductase, fumarase, malate dehydrogenase, NAD(P)H:ferredoxinoxidoreductase, carbon monoxide dehydrogenase, and hydrogenase.Specifically, the reducing equivalents extracted from CO and/or H₂ bycarbon monoxide dehydrogenase and hydrogenase are utilized to fix CO₂via the reductive TCA cycle into acetyl-CoA or acetate. Acetate can beconverted to acetyl-CoA by enzymes such as acetyl-CoA transferase,acetate kinase/phosphotransacetylase, and acetyl-CoA synthetase.Acetyl-CoA can be converted to methacrylic acid precursors, methacrylateester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate. for example,glyceraldehyde-3-phosphate, phosphoenolpyruvate, and pyruvate, bypyruvate:ferredoxin oxidoreductase and the enzymes of gluconeogenesis.Following the teachings and guidance provided herein for introducing asufficient number of encoding nucleic acids to generate a methacrylicacid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate pathway, those skilled in the art will understandthat the same engineering design also can be performed with respect tointroducing at least the nucleic acids encoding the reductive TCApathway enzymes or proteins absent in the host organism. Therefore,introduction of one or more encoding nucleic acids into the microbialorganisms of the invention such that the modified organism contains thecomplete reductive TCA pathway will confer syngas utilization ability.

Accordingly, given the teachings and guidance provided herein, thoseskilled in the art will understand that a non-naturally occurringmicrobial organism can be produced that secretes the biosynthesizedcompounds of the invention when grown on a carbon source such as acarbohydrate. Such compounds include, for example, methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate andany of the intermediate metabolites in the methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyratepathway. All that is required is to engineer in one or more of therequired enzyme or protein activities to achieve biosynthesis of thedesired compound or intermediate including, for example, inclusion ofsome or all of the methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate biosynthetic pathways.Accordingly, the invention provides a non-naturally occurring microbialorganism that produces and/or secretes methacrylic acid, methacrylateester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate when grown on acarbohydrate or other carbon source and produces and/or secretes any ofthe intermediate metabolites shown in the methacrylic acid, methacrylateester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate pathway whengrown on a carbohydrate or other carbon source. The methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrateproducing microbial organisms of the invention can initiate synthesisfrom an intermediate, for example, citramalyl-CoA, itaconyl-CoA,itaconate, citramalate, mesaconate or citraconate.

The non-naturally occurring microbial organisms of the invention areconstructed using methods well known in the art as exemplified herein toexogenously express at least one nucleic acid encoding a methacrylicacid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate pathway enzyme or protein in sufficient amounts toproduce methacrylic acid, methacrylate ester, 3-hydroxyisobutyrateand/or 2-hydroxyisobutyrate. It is understood that the microbialorganisms of the invention are cultured under conditions sufficient toproduce methacrylic acid, methacrylate ester, 3-hydroxyisobutyrateand/or 2-hydroxyisobutyrate. Following the teachings and guidanceprovided herein, the non-naturally occurring microbial organisms of theinvention can achieve biosynthesis of methacrylic acid, methacrylateester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate resulting inintracellular concentrations between about 0.1-200 mM or more.Generally, the intracellular concentration of methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate isbetween about 3-150 mM, particularly between about 5-125 mM and moreparticularly between about 8-100 mM, including about 10 mM, 20 mM, 50mM, 80 mM, or more. Intracellular concentrations between and above eachof these exemplary ranges also can be achieved from the non-naturallyoccurring microbial organisms of the invention.

In some embodiments, culture conditions include anaerobic orsubstantially anaerobic growth or maintenance conditions. Exemplaryanaerobic conditions have been described previously and are well knownin the art. Exemplary anaerobic conditions for fermentation processesare described herein and are described, for example, in U.S. publication2009/0047719, filed Aug. 10, 2007. Any of these conditions can beemployed with the non-naturally occurring microbial organisms as well asother anaerobic conditions well known in the art. Under such anaerobicor substantially anaerobic conditions, the methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrateproducers can synthesize methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate at intracellularconcentrations of 5-10 mM or more as well as all other concentrationsexemplified herein. It is understood that, even though the abovedescription refers to intracellular concentrations, methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrateproducing microbial organisms can produce methacrylic acid, methacrylateester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate intracellularlyand/or secrete the product into the culture medium.

In addition to the culturing and fermentation conditions disclosedherein, growth condition for achieving biosynthesis of methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate caninclude the addition of an osmoprotectant to the culturing conditions.In certain embodiments, the non-naturally occurring microbial organismsof the invention can be sustained, cultured or fermented as describedherein in the presence of an osmoprotectant. Briefly, an osmoprotectantrefers to a compound that acts as an osmolyte and helps a microbialorganism as described herein survive osmotic stress. Osmoprotectantsinclude, but are not limited to, betaines, amino acids, and the sugartrehalose. Non-limiting examples of such are glycine betaine, pralinebetaine, dimethylthetin, dimethylslfonioproprionate,3-dimethylsulfonio-2-methylproprionate, pipecolic acid,dimethylsulfonioacetate, choline, L-carnitine and ectoine. In oneaspect, the osmoprotectant is glycine betaine. It is understood to oneof ordinary skill in the art that the amount and type of osmoprotectantsuitable for protecting a microbial organism described herein fromosmotic stress will depend on the microbial organism used. The amount ofosmoprotectant in the culturing conditions can be, for example, no morethan about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM,no more than about 1.5 mM, no more than about 2.0 mM, no more than about2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no morethan about 7.0 mM, no more than about 10 mM, no more than about 50 mM,no more than about 100 mM or no more than about 500 mM.

In some embodiments, the carbon feedstock and other cellular uptakesources such as phosphate, ammonia, sulfate, chloride and other halogenscan be chosen to alter the isotopic distribution of the atoms present inmethacrylic acid, methacrylate este, methyl methacrylate,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate or any methacrylicacid, methacrylate este, methyl methacrylate, 3-hydroxyisobutyrateand/or 2-hydroxyisobutyrate pathway intermediate. The various carbonfeedstock and other uptake sources enumerated above will be referred toherein, collectively, as “uptake sources.” Uptake sources can provideisotopic enrichment for any atom present in the product methacrylicacid, methacrylate este, methyl methacrylate, 3-hydroxyisobutyrateand/or 2-hydroxyisobutyrate pathway intermediate, or for side productsgenerated in reactions diverging away from a methacrylic acid,methacrylate este, methyl methacrylate, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate pathway. Isotopic enrichment can be achieved forany target atom including, for example, carbon, hydrogen, oxygen,nitrogen, sulfur, phosphorus, chloride or other halogens.

In some embodiments, the uptake sources can be selected to alter thecarbon-12, carbon-13, and carbon-14 ratios. In some embodiments, theuptake sources can be selected to alter the oxygen-16, oxygen-17, andoxygen-18 ratios. In some embodiments, the uptake sources can beselected to alter the hydrogen, deuterium, and tritium ratios. In someembodiments, the uptake sources can be selected to alter the nitrogen-14and nitrogen-15 ratios. In some embodiments, the uptake sources can beselected to alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35ratios. In some embodiments, the uptake sources can be selected to alterthe phosphorus-31, phosphorus-32, and phosphorus-33 ratios. In someembodiments, the uptake sources can be selected to alter thechlorine-35, chlorine-36, and chlorine-37 ratios.

In some embodiments, the isotopic ratio of a target atom can be variedto a desired ratio by selecting one or more uptake sources. An uptakesource can be derived from a natural source, as found in nature, or froma man-made source, and one skilled in the art can select a naturalsource, a man-made source, or a combination thereof, to achieve adesired isotopic ratio of a target atom. An example of a man-made uptakesource includes, for example, an uptake source that is at leastpartially derived from a chemical synthetic reaction. Such isotopicallyenriched uptake sources can be purchased commercially or prepared in thelaboratory and/or optionally mixed with a natural source of the uptakesource to achieve a desired isotopic ratio. In some embodiments, atarget atom isotopic ratio of an uptake source can be achieved byselecting a desired origin of the uptake source as found in nature. Forexample, as discussed herein, a natural source can be a biobased derivedfrom or synthesized by a biological organism or a source such aspetroleum-based products or the atmosphere. In some such embodiments, asource of carbon, for example, can be selected from a fossilfuel-derived carbon source, which can be depleted of carbon-14, or anenvironmental or atmospheric carbon source, such as CO₂, which canpossess a larger amount of carbon-14 than its petroleum-derivedcounterpart.

The unstable carbon isotope carbon-14 or radiocarbon makes up forroughly 1 in 10¹² carbon atoms in the earth's atmosphere and has ahalf-life of about 5700 years. The stock of carbon is replenished in theupper atmosphere by a nuclear reaction involving cosmic rays andordinary nitrogen (¹⁴N). Fossil fuels contain no carbon-14, as itdecayed long ago. Burning of fossil fuels lowers the atmosphericcarbon-14 fraction, the so-called “Suess effect”.

Methods of determining the isotopic ratios of atoms in a compound arewell known to those skilled in the art. Isotopic enrichment is readilyassessed by mass spectrometry using techniques known in the art such asaccelerated mass spectrometry (AMS), Stable Isotope Ratio MassSpectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation byNuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques canbe integrated with separation techniques such as liquid chromatography(LC), high performance liquid chromatography (HPLC) and/or gaschromatography, and the like.

In the case of carbon, ASTM D6866 was developed in the United States asa standardized analytical method for determining the biobased content ofsolid, liquid, and gaseous samples using radiocarbon dating by theAmerican Society for Testing and Materials (ASTM) International. Thestandard is based on the use of radiocarbon dating for the determinationof a product's biobased content. ASTM D6866 was first published in 2004,and the current active version of the standard is ASTM D6866-11(effective Apr. 1, 2011). Radiocarbon dating techniques are well knownto those skilled in the art, including those described herein.

The biobased content of a compound is estimated by the ratio ofcarbon-14 (¹⁴C) to carbon-12 (¹²C). Specifically, the Fraction Modern(Fm) is computed from the expression: Fm=(S−B)/(M−B), where B, S and Mrepresent the ¹⁴C/¹²C ratios of the blank, the sample and the modernreference, respectively. Fraction Modern is a measurement of thedeviation of the ¹⁴C/¹²C ratio of a sample from “Modern.” Modern isdefined as 95% of the radiocarbon concentration (in AD 1950) of NationalBureau of Standards (NBS) Oxalic Acid I (i.e., standard referencematerials (SRM) 4990b) normalized to δ¹³C_(VPDB)=−19 per mil (Olsson,The use of Oxalic acid as a Standard. In, Radiocarbon Variations andAbsolute Chronology, Nobel Symposium, 12th Proc., John Wiley & Sons, NewYork (1970)). Mass spectrometry results, for example, measured by ASM,are calculated using the internationally agreed upon definition of 0.95times the specific activity of NBS Oxalic Acid I (SRM 4990b) normalizedto δ¹³C_(VPDB)=−19 per mil. This is equivalent to an absolute (AD 1950)¹⁴C/¹²C ratio of 1.176±0.010×10⁻¹² (Karlen et al., Arkiv Geoftsik,4:465-471 (1968)). The standard calculations take into account thedifferential uptake of one isotope with respect to another, for example,the preferential uptake in biological systems of C¹² over C¹³ over C¹⁴,and these corrections are reflected as a Fm corrected for δ¹³.

An oxalic acid standard (SRM 4990b or HOx 1) was made from a crop of1955 sugar beet. Although there were 1000 lbs made, this oxalic acidstandard is no longer commercially available. The Oxalic Acid IIstandard (HOx 2; N.I.S.T designation SRM 4990 C) was made from a crop of1977 French beet molasses. In the early 1980's, a group of 12laboratories measured the ratios of the two standards. The ratio of theactivity of Oxalic acid II to 1 is 1.2933±0.001 (the weighted mean). Theisotopic ratio of HOx II is −17.8 per mille. ASTM D6866-11 suggests useof the available Oxalic Acid II standard SRM 4990 C (Hox2) for themodern standard (see discussion of original vs. currently availableoxalic acid standards in Mann, Radiocarbon, 25(2):519-527 (1983)). AFm=0% represents the entire lack of carbon-14 atoms in a material, thusindicating a fossil (for example, petroleum based) carbon source. AFm=100%, after correction for the post-1950 injection of carbon-14 intothe atmosphere from nuclear bomb testing, indicates an entirely moderncarbon source. As described herein, such a “modern” source includesbiobased sources.

As described in ASTM D6866, the percent modern carbon (pMC) can begreater than 100% because of the continuing but diminishing effects ofthe 1950s nuclear testing programs, which resulted in a considerableenrichment of carbon-14 in the atmosphere as described in ASTM D6866-11.Because all sample carbon-14 activities are referenced to a “pre-bomb”standard, and because nearly all new biobased products are produced in apost-bomb environment, all pMC values (after correction for isotopicfraction) must be multiplied by 0.95 (as of 2010) to better reflect thetrue biobased content of the sample. A biobased content that is greaterthan 103% suggests that either an analytical error has occurred, or thatthe source of biobased carbon is more than several years old.

ASTM D6866 quantifies the biobased content relative to the material'stotal organic content and does not consider the inorganic carbon andother non-carbon containing substances present. For example, a productthat is 50% starch-based material and 50% water would be considered tohave a Biobased Content=100% (50% organic content that is 100% biobased)based on ASTM D6866. In another example, a product that is 50%starch-based material, 25% petroleum-based, and 25% water would have aBiobased Content=66.7% (75% organic content but only 50% of the productis biobased). In another example, a product that is 50% organic carbonand is a petroleum-based product would be considered to have a BiobasedContent=0% (50% organic carbon but from fossil sources). Thus, based onthe well known methods and known standards for determining the biobasedcontent of a compound or material, one skilled in the art can readilydetermine the biobased content and/or prepared downstream products thatutilize of the invention having a desired biobased content.

Applications of carbon-14 dating techniques to quantify bio-basedcontent of materials are known in the art (Currie et al., NuclearInstruments and Methods in Physics Research B, 172:281-287 (2000)). Forexample, carbon-14 dating has been used to quantify bio-based content interephthalate-containing materials (Colonna et al., Green Chemistry,13:2543-2548 (2011)). Notably, polypropylene terephthalate (PPT)polymers derived from renewable 1,3-propanediol and petroleum-derivedterephthalic acid resulted in Fm values near 30% (i.e., since 3/11 ofthe polymeric carbon derives from renewable 1,3-propanediol and 8/11from the fossil end member terephthalic acid) (Currie et al., supra,2000). In contrast, polybutylene terephthalate polymer derived from bothrenewable 1,4-butanediol and renewable terephthalic acid resulted inbio-based content exceeding 90% (Colonna et al., supra, 2011).

Accordingly, in some embodiments, the present invention providesmethacrylic acid, methacrylate ester, methyl methacrylate,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate or a methacrylic acid,methacrylate ester, methyl methacrylate, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate pathway intermediate that has a carbon-12,carbon-13, and carbon-14 ratio that reflects an atmospheric carbon, alsoreferred to as environmental carbon, uptake source. For example, in someaspects the methacrylic acid, methacrylate ester, methyl methacrylate,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate or a methacrylic acid,methacrylate ester, methyl methacrylate, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate intermediate can have an Fm value of at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98% or as much as 100%. In some suchembodiments, the uptake source is CO₂. In some embodiments, the presentinvention provides methacrylic acid, methacrylate ester, methylmethacrylate, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate or amethacrylic acid, methacrylate ester, methyl methacrylate,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate intermediate that has acarbon-12, carbon-13, and carbon-14 ratio that reflects petroleum-basedcarbon uptake source. In this aspect, the methacrylic acid, methacrylateester, methyl methacrylate, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate or a methacrylic acid, methacrylate ester, methylmethacrylate, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrateintermediate can have an Fm value of less than 95%, less than 90%, lessthan 85%, less than 80%, less than 75%, less than 70%, less than 65%,less than 60%, less than 55%, less than 50%, less than 45%, less than40%, less than 35%, less than 30%, less than 25%, less than 20%, lessthan 15%, less than 10%, less than 5%, less than 2% or less than 1%. Insome embodiments, the present invention provides methacrylic acid,methacrylate ester, methyl methacrylate, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate or a methacrylic acid, methacrylate ester, methylmethacrylate, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrateintermediate that has a carbon-12, carbon-13, and carbon-14 ratio thatis obtained by a combination of an atmospheric carbon uptake source witha petroleum-based uptake source. Using such a combination of uptakesources is one way by which the carbon-12, carbon-13, and carbon-14ratio can be varied, and the respective ratios would reflect theproportions of the uptake sources.

Further, the present invention relates to the biologically producedmethacrylic acid, methacrylate ester, methyl methacrylate,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate or methacrylic acid,methacrylate ester, methyl methacrylate, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate intermediate as disclosed herein, and to theproducts derived therefrom, wherein the methacrylic acid, methacrylateester, methyl methacrylate, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate or a methacrylic acid, methacrylate ester, methylmethacrylate, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrateintermediate has a carbon-12, carbon-13, and carbon-14 isotope ratio ofabout the same value as the CO₂ that occurs in the environment. Forexample, in some aspects the invention provides bioderived methacrylicacid, methacrylate ester, methyl methacrylate, 3-hydroxyisobutyrateand/or 2-hydroxyisobutyrate or a bioderived methacrylic acid,methacrylate ester, methyl methacrylate, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate intermediate having a carbon-12 versus carbon-13versus carbon-14 isotope ratio of about the same value as the CO₂ thatoccurs in the environment, or any of the other ratios disclosed herein.It is understood, as disclosed herein, that a product can have acarbon-12 versus carbon-13 versus carbon-14 isotope ratio of about thesame value as the CO₂ that occurs in the environment, or any of theratios disclosed herein, wherein the product is generated frombioderived methacrylic acid, methacrylate ester, methyl methacrylate,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate or a bioderivedmethacrylic acid, methacrylate ester, methyl methacrylate,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate intermediate asdisclosed herein, wherein the bioderived product is chemically modifiedto generate a final product. Methods of chemically modifying abioderived product of methacrylic acid, methacrylate ester, methylmethacrylate, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate, or anintermediate thereof, to generate a desired product are well known tothose skilled in the art, as described herein. The invention furtherprovides polymers, including polymethyl methacrylate (PMMA), acrylicplastics, and co-polymers, such as the co-polymer methylmethacrylate-butadiene-styrene (MBS), and the like, having a carbon-12versus carbon-13 versus carbon-14 isotope ratio of about the same valueas the CO₂ that occurs in the environment, wherein the polymers,including polymethyl methacrylate, acrylic plastics and the co-polymermethyl methacrylate-butadiene-styrene (MBS), are generated directly fromor in combination with bioderived methacrylic acid, methacrylate ester,methyl methacrylate, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate ora bioderived methacrylic acid, methacrylate ester, methyl methacrylate,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate intermediate asdisclosed herein.

Methacrylic acid, methacrylate ester, methyl methacrylate,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate are chemicals used incommercial and industrial applications. Non-limiting examples of suchapplications include production of polymers, including polymethylmethacrylate (PMMA), also referred to as acrylic glass, Lucite™ orPlexiglas™, acrylic plastics and co-polymers, such as the co-polymermethyl methacrylate-butadiene-styrene (MBS). Moreover, methacrylic acid,methacrylate ester, methyl methacrylate, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate are also used as raw materials in the production ofa wide range of products including polymers, including polymethylmethacrylate acrylic plastics and the co-polymer methylmethacrylate-butadiene-styrene (MBS). Accordingly, in some embodiments,the invention provides biobased polymers, including polymethylmethacrylate, acrylic plastics, and co-polymers, including methylmethacrylate-butadiene-styrene (MBS), comprising one or more bioderivedmethacrylic acid, methacrylate ester, methyl methacrylate,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate or bioderivedmethacrylic acid, methacrylate ester, methyl methacrylate,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate intermediate producedby a non-naturally occurring microorganism of the invention or producedusing a method disclosed herein. Such polymers, including polymethylmethacrylate, acrylic plastics, and co-polymers, including methylmethacrylate-butadiene-styrene, can have a mixture of bioderived andpetroleum based precursors, in desired ratios as described above.

As used herein, the term “bioderived” means derived from or synthesizedby a biological organism and can be considered a renewable resourcesince it can be generated by a biological organism. Such a biologicalorganism, in particular the microbial organisms of the inventiondisclosed herein, can utilize feedstock or biomass, such as, sugars orcarbohydrates obtained from an agricultural, plant, bacterial, or animalsource. Alternatively, the biological organism can utilize atmosphericcarbon As used herein, the term “biobased” means a product as describedabove that is composed, in whole or in part, of a bioderived compound ofthe invention. A biobased or bioderived product is in contrast to apetroleum derived product, wherein such a product is derived from orsynthesized from petroleum or a petrochemical feedstock.

In some embodiments, the invention provides polymers, includingpolymethyl methacrylate acrylic plastics and the co-polymer methylmethacrylate-butadiene-styrene (MBS), comprising bioderived methacrylicacid, methacrylate ester, methyl methacrylate, 3-hydroxyisobutyrateand/or 2-hydroxyisobutyrate or bioderived methacrylic acid, methacrylateester, methyl methacrylate, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate intermediate, wherein the bioderived methacrylicacid, methacrylate ester, methyl methacrylate, 3-hydroxyisobutyrateand/or 2-hydroxyisobutyrate or bioderived methacrylic acid, methacrylateester, methyl methacrylate, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate intermediate includes all or part of themethacrylic acid, methacrylate ester, methyl methacrylate,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate or methacrylic acid,methacrylate ester, methyl methacrylate, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate intermediate used in the production of polymers,including polymethyl methacrylate acrylic plastics and the co-polymermethyl methacrylate-butadiene-styrene (MBS). Thus, in some aspects, theinvention provides a biobased polymers, including polymethylmethacrylate acrylic plastics and the co-polymer methylmethacrylate-butadiene-styrene (MBS), comprising at least 2%, at least3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98% or100% bioderived methacrylic acid, methacrylate ester, methylmethacrylate, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate orbioderived methacrylic acid, methacrylate ester, methyl methacrylate,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate intermediate asdisclosed herein. Additionally, in some aspects, the invention providesa biobased polymers, including polymethyl methacrylate acrylic plasticsand the co-polymer methyl methacrylate-butadiene-styrene (MBS), whereinthe methacrylic acid, methacrylate ester, methyl methacrylate,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate or methacrylic acid,methacrylate ester, methyl methacrylate, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate intermediate used in its production is acombination of bioderived and petroleum derived methacrylic acid,methacrylate ester, methyl methacrylate, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate or methacrylic acid, methacrylate ester, methylmethacrylate, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrateintermediate. For example, a biobased polymers, including polymethylmethacrylate acrylic plastics and the co-polymer methylmethacrylate-butadiene-styrene (MBS), can be produced using 50%bioderived methacrylic acid, methacrylate ester, methyl methacrylate,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate and 50% petroleumderived methacrylic acid, methacrylate ester such as methylmethacrylate, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate, or otherdesired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%,100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleumderived precursors, so long as at least a portion of the productcomprises a bioderived product produced by the microbial organismsdisclosed herein. It is understood that methods for producing polymers,including polymethyl methacrylate acrylic plastics and the co-polymermethyl methacrylate-butadiene-styrene (MBS), using the bioderivedmethacrylic acid, methacrylate ester, methyl methacrylate,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate or bioderivedmethacrylic acid, methacrylate ester, methyl methacrylate,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate intermediate of theinvention are well known in the art.

The culture conditions can include, for example, liquid cultureprocedures as well as fermentation and other large scale cultureprocedures. As described herein, particularly useful yields of thebiosynthetic products of the invention can be obtained under anaerobicor substantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achievingbiosynthesis of methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate includes anaerobicculture or fermentation conditions. In certain embodiments, thenon-naturally occurring microbial organisms of the invention can besustained, cultured or fermented under anaerobic or substantiallyanaerobic conditions. Briefly, anaerobic conditions refers to anenvironment devoid of oxygen. Substantially anaerobic conditionsinclude, for example, a culture, batch fermentation or continuousfermentation such that the dissolved oxygen concentration in the mediumremains between 0 and 10% of saturation. Substantially anaerobicconditions also includes growing or resting cells in liquid medium or onsolid agar inside a sealed chamber maintained with an atmosphere of lessthan 1% oxygen. The percent of oxygen can be maintained by, for example,sparging the culture with an N₂/CO₂ mixture or other suitable non-oxygengas or gases.

The culture conditions described herein can be scaled up and growncontinuously for manufacturing of methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate. Exemplary growthprocedures include, for example, fed-batch fermentation and batchseparation; fed-batch fermentation and continuous separation, orcontinuous fermentation and continuous separation. All of theseprocesses are well known in the art. Fermentation procedures areparticularly useful for the biosynthetic production of commercialquantities of methacrylic acid, methacrylate ester, 3-hydroxyisobutyrateand/or 2-hydroxyisobutyrate. Generally, and as with non-continuousculture procedures, the continuous and/or near-continuous production ofmethacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate will include culturing a non-naturally occurringmethacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate producing organism of the invention in sufficientnutrients and medium to sustain and/or nearly sustain growth in anexponential phase. Continuous culture under such conditions can include,for example, growth for 1 day, 2, 3, 4, 5, 6 or 7 days or more.Additionally, continuous culture can include longer time periods of 1week, 2, 3, 4 or 5 or more weeks and up to several months.Alternatively, organisms of the invention can be cultured for hours, ifsuitable for a particular application. It is to be understood that thecontinuous and/or near-continuous culture conditions also can includeall time intervals in between these exemplary periods. It is furtherunderstood that the time of culturing the microbial organism of theinvention is for a sufficient period of time to produce a sufficientamount of product for a desired purpose.

Fermentation procedures are well known in the art. Briefly, fermentationfor the biosynthetic production of methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate can be utilized in, forexample, fed-batch fermentation and batch separation; fed-batchfermentation and continuous separation, or continuous fermentation andcontinuous separation. Examples of batch and continuous fermentationprocedures are well known in the art.

In addition to the above fermentation procedures using the methacrylicacid, methacrylate ester, methyl methacrylate, 3-hydroxyisobutyrate,3-hydroxyisobutyrate ester, 2-hydroxyisobutyrate and/or2-hydroxyisobutyrate ester producers of the invention for continuousproduction of substantial quantities of methacrylic acid, methacrylateester, methyl methacrylate, 3-hydroxyisobutyrate, 3-hydroxyisobutyrateester, 2-hydroxyisobutyrate and/or 2-hydroxyisobutyrate ester producersalso can be, for example, simultaneously subjected to chemical synthesisprocedures to convert the product to other compounds or the product canbe separated from the fermentation culture and sequentially subjected tochemical or enzymatic conversion to convert the product to othercompounds, if desired. For example, in some embodiments, the inventionprovides chemical dehydration of a 3-hydroxyisobutyrate ester such asmethyl-3-hydroxyisobutyrate or a 2-hydroxyisobutyrate ester such asmethyl-2-hydroxyisobutyrate to a methacrylate ester such as methylmethacrylate. It is understood that methods and chemical compounds forcatalyzing the dehydration of such compounds are well known in the art.

3-hydroxyisobutyrate esters and 2-hydroxyisobutyrate esters can bechemically dehydrated with formation of methacrylate esters, startingwith pure 3-hydroxyisobutyrate or 2-hydroxyisobutyrate isolated from thefermentation solution or starting with aqueous or organic solutions of3-hydroxyisobutyrate ester or 2-hydroxyisobutyrate ester, isolated inwork up of the fermentation solution. Such solutions of3-hydroxyisobutyrate ester or 2-hydroxyisobutyrate ester can also beconcentrated before the dehydration step, for example by means ofdistillation, optionally in the presence of a suitable entrainer.

The dehydration reaction can be carried out in liquid phase or in thegas phase. The dehydration reaction can be carried out in the presenceof a catalyst, the nature of the catalyst employed depending on whethera gas-phase or a liquid-phase reaction is carried out.

Suitable dehydration catalysts include both acidic catalysts andalkaline catalysts. Acidic catalysts, in particular can exhibit adecreased tendency to form oligomers. The dehydration catalyst can beemployed as a homogeneous catalyst, a heterogeneous catalyst, orcombinations thereof. Heterogeneous catalysts can be used in conjunctionwith a suitable support material. Such a support can itself be acidic oralkaline and provide the acidic or alkaline dehydration catalyst or acatalyst can be applied to an inert support.

Suitable supports which serve as dehydration catalysts include naturalor synthetic silicates such as mordenite, montmorillonite, acidiczeolites; supports which are coated with monobasic, dibasic or polybasicinorganic acids, such as phosphoric acid, or with acidic salts ofinorganic acids, such as oxides or silicates, for example Al2O3, TiO2;oxides and mixed oxides such as γ-Al2O3 and ZnO—Al2O3 mixed oxides ofheteropolyacids. Alkaline substances which act both as dehydrationcatalyst and as a support a support material include alkali, alkalineearth, lanthanum, lanthoids or a combinations thereof as their oxides. Afurther class of materials that can effect dehydration are ionexchangers which can be used in either alkaline or acidic form.

Suitable homogeneous dehydration catalysts include inorganic acids, suchas phosphorus-containing acids such as phosphoric acid. Inorganic acidscan be immobilized on the support material by immersion or impregnation.

In some embodiments, dehydration reaction is carried out in the gasphase using conventional apparatuses known in the art, for exampletubular reactors, shell-and-tube heat exchangers and reactors whichcomprise thermoplates as heat exchangers. In some embodiments, gas-phasedehydration can utilize isolated 3-hydroxyisobutyrate esters orsolutions of the ester, the ester being introduced into a reactor withfixed-bed catalysts. Thermal dehydration in the liquid phase can becarried out in a temperature range of between 200° C. and 350° C., andin some embodiments between 250 and 300° C.

To generate better producers, metabolic modeling can be utilized tooptimize growth conditions. Modeling can also be used to design geneknockouts that additionally optimize utilization of the pathway (see,for example, U.S. patent publications US 2002/0012939, US 2003/0224363,US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 andUS 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allowsreliable predictions of the effects on cell growth of shifting themetabolism towards more efficient production of methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate.

One computational method for identifying and designing metabolicalterations favoring biosynthesis of a desired product is the OptKnockcomputational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657(2003)). OptKnock is a metabolic modeling and simulation program thatsuggests gene deletion or disruption strategies that result ingenetically stable microorganisms which overproduce the target product.Specifically, the framework examines the complete metabolic and/orbiochemical network of a microorganism in order to suggest geneticmanipulations that force the desired biochemical to become an obligatorybyproduct of cell growth. By coupling biochemical production with cellgrowth through strategically placed gene deletions or other functionalgene disruption, the growth selection pressures imposed on theengineered strains after long periods of time in a bioreactor lead toimprovements in performance as a result of the compulsory growth-coupledbiochemical production. Lastly, when gene deletions are constructedthere is a negligible possibility of the designed strains reverting totheir wild-type states because the genes selected by OptKnock are to becompletely removed from the genome. Therefore, this computationalmethodology can be used to either identify alternative pathways thatlead to biosynthesis of a desired product or used in connection with thenon-naturally occurring microbial organisms for further optimization ofbiosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computationalmethod and system for modeling cellular metabolism. The OptKnock programrelates to a framework of models and methods that incorporate particularconstraints into flux balance analysis (FBA) models. These constraintsinclude, for example, qualitative kinetic information, qualitativeregulatory information, and/or DNA microarray experimental data.OptKnock also computes solutions to various metabolic problems by, forexample, tightening the flux boundaries derived through flux balancemodels and subsequently probing the performance limits of metabolicnetworks in the presence of gene additions or deletions. OptKnockcomputational framework allows the construction of model formulationsthat allow an effective query of the performance limits of metabolicnetworks and provides methods for solving the resulting mixed-integerlinear programming problems. The metabolic modeling and simulationmethods referred to herein as OptKnock are described in, for example,U.S. publication 2002/0168654, filed Jan. 10, 2002, in InternationalPatent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S. publication2009/0047719, filed Aug. 10, 2007.

Another computational method for identifying and designing metabolicalterations favoring biosynthetic production of a product is a metabolicmodeling and simulation system termed SimPheny®. This computationalmethod and system is described in, for example, U.S. publication2003/0233218, filed Jun. 14, 2002, and in International PatentApplication No. PCT/US03/18838, filed Jun. 13, 2003. SimPheny® is acomputational system that can be used to produce a network model insilico and to simulate the flux of mass, energy or charge through thechemical reactions of a biological system to define a solution spacethat contains any and all possible functionalities of the chemicalreactions in the system, thereby determining a range of allowedactivities for the biological system. This approach is referred to asconstraints-based modeling because the solution space is defined byconstraints such as the known stoichiometry of the included reactions aswell as reaction thermodynamic and capacity constraints associated withmaximum fluxes through reactions. The space defined by these constraintscan be interrogated to determine the phenotypic capabilities andbehavior of the biological system or of its biochemical components.

These computational approaches are consistent with biological realitiesbecause biological systems are flexible and can reach the same result inmany different ways. Biological systems are designed throughevolutionary mechanisms that have been restricted by fundamentalconstraints that all living systems must face. Therefore,constraints-based modeling strategy embraces these general realities.Further, the ability to continuously impose further restrictions on anetwork model via the tightening of constraints results in a reductionin the size of the solution space, thereby enhancing the precision withwhich physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in theart will be able to apply various computational frameworks for metabolicmodeling and simulation to design and implement biosynthesis of adesired compound in host microbial organisms. Such metabolic modelingand simulation methods include, for example, the computational systemsexemplified above as SimPheny® and OptKnock. For illustration of theinvention, some methods are described herein with reference to theOptKnock computation framework for modeling and simulation. Thoseskilled in the art will know how to apply the identification, design andimplementation of the metabolic alterations using OptKnock to any ofsuch other metabolic modeling and simulation computational frameworksand methods well known in the art.

The methods described above will provide one set of metabolic reactionsto disrupt. Elimination of each reaction within the set or metabolicmodification can result in a desired product as an obligatory productduring the growth phase of the organism. Because the reactions areknown, a solution to the bilevel OptKnock problem also will provide theassociated gene or genes encoding one or more enzymes that catalyze eachreaction within the set of reactions. Identification of a set ofreactions and their corresponding genes encoding the enzymesparticipating in each reaction is generally an automated process,accomplished through correlation of the reactions with a reactiondatabase having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in orderto achieve production of a desired product are implemented in the targetcell or organism by functional disruption of at least one gene encodingeach metabolic reaction within the set. One particularly useful means toachieve functional disruption of the reaction set is by deletion of eachencoding gene. However, in some instances, it can be beneficial todisrupt the reaction by other genetic aberrations including, forexample, mutation, deletion of regulatory regions such as promoters orcis binding sites for regulatory factors, or by truncation of the codingsequence at any of a number of locations. These latter aberrations,resulting in less than total deletion of the gene set can be useful, forexample, when rapid assessments of the coupling of a product are desiredor when genetic reversion is less likely to occur.

To identify additional productive solutions to the above describedbilevel OptKnock problem which lead to further sets of reactions todisrupt or metabolic modifications that can result in the biosynthesis,including growth-coupled biosynthesis of a desired product, anoptimization method, termed integer cuts, can be implemented. Thismethod proceeds by iteratively solving the OptKnock problem exemplifiedabove with the incorporation of an additional constraint referred to asan integer cut at each iteration. Integer cut constraints effectivelyprevent the solution procedure from choosing the exact same set ofreactions identified in any previous iteration that obligatorily couplesproduct biosynthesis to growth. For example, if a previously identifiedgrowth-coupled metabolic modification specifies reactions 1, 2, and 3for disruption, then the following constraint prevents the samereactions from being simultaneously considered in subsequent solutions.The integer cut method is well known in the art and can be founddescribed in, for example, Burgard et al., Biotechnol. Prog. 17:791-797(2001). As with all methods described herein with reference to their usein combination with the OptKnock computational framework for metabolicmodeling and simulation, the integer cut method of reducing redundancyin iterative computational analysis also can be applied with othercomputational frameworks well known in the art including, for example,SimPheny®.

The methods exemplified herein allow the construction of cells andorganisms that biosynthetically produce a desired product, including theobligatory coupling of production of a target biochemical product togrowth of the cell or organism engineered to harbor the identifiedgenetic alterations. Therefore, the computational methods describedherein allow the identification and implementation of metabolicmodifications that are identified by an in silico method selected fromOptKnock or SimPheny®. The set of metabolic modifications can include,for example, addition of one or more biosynthetic pathway enzymes and/orfunctional disruption of one or more metabolic reactions including, forexample, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on thepremise that mutant microbial networks can be evolved towards theircomputationally predicted maximum-growth phenotypes when subjected tolong periods of growth selection. In other words, the approach leveragesan organism's ability to self-optimize under selective pressures. TheOptKnock framework allows for the exhaustive enumeration of genedeletion combinations that force a coupling between biochemicalproduction and cell growth based on network stoichiometry. Theidentification of optimal gene/reaction knockouts requires the solutionof a bilevel optimization problem that chooses the set of activereactions such that an optimal growth solution for the resulting networkoverproduces the biochemical of interest (Burgard et al., Biotechnol.Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employedto identify essential genes for metabolic pathways as exemplifiedpreviously and described in, for example, U.S. patent publications US2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No.7,127,379. As disclosed herein, the OptKnock mathematical framework canbe applied to pinpoint gene deletions leading to the growth-coupledproduction of a desired product. Further, the solution of the bilevelOptKnock problem provides only one set of deletions. To enumerate allmeaningful solutions, that is, all sets of knockouts leading togrowth-coupled production formation, an optimization technique, termedinteger cuts, can be implemented. This entails iteratively solving theOptKnock problem with the incorporation of an additional constraintreferred to as an integer cut at each iteration, as discussed above.

As disclosed herein, a nucleic acid encoding a desired activity of amethacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate pathway can be introduced into a host organism. Insome cases, it can be desirable to modify an activity of a methacrylicacid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate pathway enzyme or protein to increase production ofmethacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate. For example, known mutations that increase theactivity of a protein or enzyme can be introduced into an encodingnucleic acid molecule. Additionally, optimization methods can be appliedto increase the activity of an enzyme or protein and/or decrease aninhibitory activity, for example, decrease the activity of a negativeregulator.

One such optimization method is directed evolution. Directed evolutionis a powerful approach that involves the introduction of mutationstargeted to a specific gene in order to improve and/or alter theproperties of an enzyme. Improved and/or altered enzymes can beidentified through the development and implementation of sensitivehigh-throughput screening assays that allow the automated screening ofmany enzyme variants (for example, >10⁴). Iterative rounds ofmutagenesis and screening typically are performed to afford an enzymewith optimized properties. Computational algorithms that can help toidentify areas of the gene for mutagenesis also have been developed andcan significantly reduce the number of enzyme variants that need to begenerated and screened. Numerous directed evolution technologies havebeen developed (for reviews, see Hibbert et al., Biomol. Eng 22:11-19(2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical andbiotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press;Otten and Quax. Biomol. Eng 22:1-9 (2005); and Sen et al., Appl Biochem.Biotechnol 143:212-223 (2007)) to be effective at creating diversevariant libraries, and these methods have been successfully applied tothe improvement of a wide range of properties across many enzymeclasses. Enzyme characteristics that have been improved and/or alteredby directed evolution technologies include, for example:selectivity/specificity, for conversion of non-natural substrates;temperature stability, for robust high temperature processing; pHstability, for bioprocessing under lower or higher pH conditions;substrate or product tolerance, so that high product titers can beachieved; binding (K_(m)), including broadening substrate binding toinclude non-natural substrates; inhibition (K_(i)), to remove inhibitionby products, substrates, or key intermediates; activity (kcat), toincreases enzymatic reaction rates to achieve desired flux; expressionlevels, to increase protein yields and overall pathway flux; oxygenstability, for operation of air sensitive enzymes under aerobicconditions; and anaerobic activity, for operation of an aerobic enzymein the absence of oxygen.

Described below in more detail are exemplary methods that have beendeveloped for the mutagenesis and diversification of genes to targetdesired properties of specific enzymes. Such methods are well known tothose skilled in the art. Any of these can be used to alter and/oroptimize the activity of a methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate pathway enzyme orprotein.

EpPCR (Pritchard et al., J Theor. Biol. 234:497-509 (2005)) introducesrandom point mutations by reducing the fidelity of DNA polymerase in PCRreactions by the addition of Mn²⁺ ions, by biasing dNTP concentrations,or by other conditional variations. The five step cloning process toconfine the mutagenesis to the target gene of interest involves: 1)error-prone PCR amplification of the gene of interest; 2) restrictionenzyme digestion; 3) gel purification of the desired DNA fragment; 4)ligation into a vector; 5) transformation of the gene variants into asuitable host and screening of the library for improved performance.This method can generate multiple mutations in a single genesimultaneously, which can be useful to screen a larger number ofpotential variants having a desired activity. A high number of mutantscan be generated by EpPCR, so a high-throughput screening assay or aselection method, for example, using robotics, is useful to identifythose with desirable characteristics.

Error-prone Rolling Circle Amplification (epRCA) (Fujii et al., NucleicAcids Res. 32:e145 (2004); and Fujii et al., Nat. Protoc. 1:2493-2497(2006)) has many of the same elements as epPCR except a whole circularplasmid is used as the template and random 6-mers with exonucleaseresistant thiophosphate linkages on the last 2 nucleotides are used toamplify the plasmid followed by transformation into cells in which theplasmid is re-circularized at tandem repeats. Adjusting the Mn²⁺concentration can vary the mutation rate somewhat. This technique uses asimple error-prone, single-step method to create a full copy of theplasmid with 3-4 mutations/kbp. No restriction enzyme digestion orspecific primers are required. Additionally, this method is typicallyavailable as a commercially available kit.

DNA or Family Shuffling (Stemmer, Proc Natl Acad Sci USA 91:10747-10751(1994)); and Stemmer, Nature 370:389-391 (1994)) typically involvesdigestion of two or more variant genes with nucleases such as Dnase I orEndoV to generate a pool of random fragments that are reassembled bycycles of annealing and extension in the presence of DNA polymerase tocreate a library of chimeric genes. Fragments prime each other andrecombination occurs when one copy primes another copy (templateswitch). This method can be used with >1 kbp DNA sequences. In additionto mutational recombinants created by fragment reassembly, this methodintroduces point mutations in the extension steps at a rate similar toerror-prone PCR. The method can be used to remove deleterious, randomand neutral mutations.

Staggered Extension (StEP) (Zhao et al., Nat. Biotechnol. 16:258-261(1998)) entails template priming followed by repeated cycles of 2 stepPCR with denaturation and very short duration of annealing/extension (asshort as 5 sec). Growing fragments anneal to different templates andextend further, which is repeated until full-length sequences are made.Template switching means most resulting fragments have multiple parents.Combinations of low-fidelity polymerases (Taq and Mutazyme) reduceerror-prone biases because of opposite mutational spectra.

In Random Priming Recombination (RPR) random sequence primers are usedto generate many short DNA fragments complementary to different segmentsof the template (Shao et al., Nucleic Acids Res 26:681-683 (1998)). Basemisincorporation and mispriming via epPCR give point mutations. ShortDNA fragments prime one another based on homology and are recombined andreassembled into full-length by repeated thermocycling. Removal oftemplates prior to this step assures low parental recombinants. Thismethod, like most others, can be performed over multiple iterations toevolve distinct properties. This technology avoids sequence bias, isindependent of gene length, and requires very little parent DNA for theapplication.

In Heteroduplex Recombination linearized plasmid DNA is used to formheteroduplexes that are repaired by mismatch repair (Volkov et al,Nucleic Acids Res. 27:e18 (1999); and Volkov et al., Methods Enzymol.328:456-463 (2000)). The mismatch repair step is at least somewhatmutagenic. Heteroduplexes transform more efficiently than linearhomoduplexes. This method is suitable for large genes and whole operons.

Random Chimeragenesis on Transient Templates (RACHITT) (Coco et al.,Nat. Biotechnol. 19:354-359 (2001)) employs Dnase I fragmentation andsize fractionation of single stranded DNA (ssDNA). Homologous fragmentsare hybridized in the absence of polymerase to a complementary ssDNAscaffold. Any overlapping unhybridized fragment ends are trimmed down byan exonuclease. Gaps between fragments are filled in and then ligated togive a pool of full-length diverse strands hybridized to the scaffold,which contains U to preclude amplification. The scaffold then isdestroyed and is replaced by a new strand complementary to the diversestrand by PCR amplification. The method involves one strand (scaffold)that is from only one parent while the priming fragments derive fromother genes, and the parent scaffold is selected against. Thus, noreannealing with parental fragments occurs. Overlapping fragments aretrimmed with an exonuclease. Otherwise, this is conceptually similar toDNA shuffling and StEP. Therefore, there should be no siblings, fewinactives, and no unshuffled parentals. This technique has advantages inthat few or no parental genes are created and many more crossovers canresult relative to standard DNA shuffling.

Recombined Extension on Truncated templates (RETT) entails templateswitching of unidirectionally growing strands from primers in thepresence of unidirectional ssDNA fragments used as a pool of templates(Lee et al., J. Molec. Catalysis 26:119-129 (2003)). No DNAendonucleases are used. Unidirectional ssDNA is made by DNA polymerasewith random primers or serial deletion with exonuclease. UnidirectionalssDNA are only templates and not primers. Random priming andexonucleases do not introduce sequence bias as true of enzymaticcleavage of DNA shuffling/RACHITT. RETT can be easier to optimize thanStEP because it uses normal PCR conditions instead of very shortextensions. Recombination occurs as a component of the PCR steps, thatis, no direct shuffling. This method can also be more random than StEPdue to the absence of pauses.

In Degenerate Oligonucleotide Gene Shuffling (DOGS) degenerate primersare used to control recombination between molecules; (Bergquist andGibbs, Methods Mol. Biol. 352:191-204 (2007); Bergquist et al., Biomol.Eng 22:63-72 (2005); Gibbs et al., Gene 271:13-20 (2001)) this can beused to control the tendency of other methods such as DNA shuffling toregenerate parental genes. This method can be combined with randommutagenesis (epPCR) of selected gene segments. This can be a good methodto block the reformation of parental sequences. No endonucleases areneeded. By adjusting input concentrations of segments made, one can biastowards a desired backbone. This method allows DNA shuffling fromunrelated parents without restriction enzyme digests and allows a choiceof random mutagenesis methods.

Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY)creates a combinatorial library with 1 base pair deletions of a gene orgene fragment of interest (Ostermeier et al., Proc. Natl. Acad. Sci. USA96:3562-3567 (1999); and Ostermeier et al., Nat. Biotechnol.17:1205-1209 (1999)). Truncations are introduced in opposite directionon pieces of 2 different genes. These are ligated together and thefusions are cloned. This technique does not require homology between the2 parental genes. When ITCHY is combined with DNA shuffling, the systemis called SCRATCHY (see below). A major advantage of both is no need forhomology between parental genes; for example, functional fusions betweenan E. coli and a human gene were created via ITCHY. When ITCHY librariesare made, all possible crossovers are captured.

Thio-Incremental Truncation for the Creation of Hybrid Enzymes(THIO-ITCHY) is similar to ITCHY except that phosphothioate dNTPs areused to generate truncations (Lutz et al., Nucleic Acids Res 29:E16(2001)). Relative to ITCHY, THIO-ITCHY can be easier to optimize,provide more reproducibility, and adjustability.

SCRATCHY combines two methods for recombining genes, ITCHY and DNAshuffling (Lutz et al., Proc. Natl. Acad. Sci. USA 98:11248-11253(2001)). SCRATCHY combines the best features of ITCHY and DNA shuffling.First, ITCHY is used to create a comprehensive set of fusions betweenfragments of genes in a DNA homology-independent fashion. Thisartificial family is then subjected to a DNA-shuffling step to augmentthe number of crossovers. Computational predictions can be used inoptimization. SCRATCHY is more effective than DNA shuffling whensequence identity is below 80%.

In Random Drift Mutagenesis (RNDM) mutations are made via epPCR followedby screening/selection for those retaining usable activity (Bergquist etal., Biomol. Eng. 22:63-72 (2005)). Then, these are used in DOGS togenerate recombinants with fusions between multiple active mutants orbetween active mutants and some other desirable parent. Designed topromote isolation of neutral mutations; its purpose is to screen forretained catalytic activity whether or not this activity is higher orlower than in the original gene. RNDM is usable in high throughputassays when screening is capable of detecting activity above background.RNDM has been used as a front end to DOGS in generating diversity. Thetechnique imposes a requirement for activity prior to shuffling or othersubsequent steps; neutral drift libraries are indicated to result inhigher/quicker improvements in activity from smaller libraries. Thoughpublished using epPCR, this could be applied to other large-scalemutagenesis methods.

Sequence Saturation Mutagenesis (SeSaM) is a random mutagenesis methodthat: 1) generates a pool of random length fragments using randomincorporation of a phosphothioate nucleotide and cleavage; this pool isused as a template to 2) extend in the presence of “universal” basessuch as inosine; 3) replication of an inosine-containing complementgives random base incorporation and, consequently, mutagenesis (Wong etal., Biotechnol. J. 3:74-82 (2008); Wong et al., Nucleic Acids Res.32:e26 (2004); and Wong et al., Anal. Biochem. 341:187-189 (2005)).Using this technique it can be possible to generate a large library ofmutants within 2 to 3 days using simple methods. This technique isnon-directed in comparison to the mutational bias of DNA polymerases.Differences in this approach makes this technique complementary (or analternative) to epPCR.

In Synthetic Shuffling, overlapping oligonucleotides are designed toencode “all genetic diversity in targets” and allow a very highdiversity for the shuffled progeny (Ness et al., Nat. Biotechnol.20:1251-1255 (2002)). In this technique, one can design the fragments tobe shuffled. This aids in increasing the resulting diversity of theprogeny. One can design sequence/codon biases to make more distantlyrelated sequences recombine at rates approaching those observed withmore closely related sequences. Additionally, the technique does notrequire physically possessing the template genes.

Nucleotide Exchange and Excision Technology NexT exploits a combinationof dUTP incorporation followed by treatment with uracil DNA glycosylaseand then piperidine to perform endpoint DNA fragmentation (Muller etal., Nucleic Acids Res. 33:e117 (2005)). The gene is reassembled usinginternal PCR primer extension with proofreading polymerase. The sizesfor shuffling are directly controllable using varying dUPT::dTTP ratios.This is an end point reaction using simple methods for uracilincorporation and cleavage. Other nucleotide analogs, such as8-oxo-guanine, can be used with this method. Additionally, the techniqueworks well with very short fragments (86 bp) and has a low error rate.The chemical cleavage of DNA used in this technique results in very fewunshuffled clones.

In Sequence Homology-Independent Protein Recombination (SHIPREC), alinker is used to facilitate fusion between two distantly related orunrelated genes. Nuclease treatment is used to generate a range ofchimeras between the two genes. These fusions result in libraries ofsingle-crossover hybrids (Sieber et al., Nat. Biotechnol. 19:456-460(2001)). This produces a limited type of shuffling and a separateprocess is required for mutagenesis. In addition, since no homology isneeded, this technique can create a library of chimeras with varyingfractions of each of the two unrelated parent genes. SHIPREC was testedwith a heme-binding domain of a bacterial CP450 fused to N-terminalregions of a mammalian CP450; this produced mammalian activity in a moresoluble enzyme.

In Gene Site Saturation Mutagenesis™ (GSSM™) the starting materials area supercoiled dsDNA plasmid containing an insert and two primers whichare degenerate at the desired site of mutations (Kretz et al., MethodsEnzymol. 388:3-11 (2004)). Primers carrying the mutation of interest,anneal to the same sequence on opposite strands of DNA. The mutation istypically in the middle of the primer and flanked on each side byapproximately 20 nucleotides of correct sequence. The sequence in theprimer is NNN or NNK (coding) and MNN (noncoding) (N=all 4, K=G, T, M=A,C). After extension, DpnI is used to digest dam-methylated DNA toeliminate the wild-type template. This technique explores all possibleamino acid substitutions at a given locus (that is, one codon). Thetechnique facilitates the generation of all possible replacements at asingle-site with no nonsense codons and results in equal to near-equalrepresentation of most possible alleles. This technique does not requireprior knowledge of the structure, mechanism, or domains of the targetenzyme. If followed by shuffling or Gene Reassembly, this technologycreates a diverse library of recombinants containing all possiblecombinations of single-site up-mutations. The usefulness of thistechnology combination has been demonstrated for the successfulevolution of over 50 different enzymes, and also for more than oneproperty in a given enzyme.

Combinatorial Cassette Mutagenesis (CCM) involves the use of shortoligonucleotide cassettes to replace limited regions with a large numberof possible amino acid sequence alterations (Reidhaar-Olson et al.Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al. Science241:53-57 (1988)). Simultaneous substitutions at two or three sites arepossible using this technique. Additionally, the method tests a largemultiplicity of possible sequence changes at a limited range of sites.This technique has been used to explore the information content of thelambda repressor DNA-binding domain.

Combinatorial Multiple Cassette Mutagenesis (CMCM) is essentiallysimilar to CCM except it is employed as part of a larger program: 1) useof epPCR at high mutation rate to 2) identify hot spots and hot regionsand then 3) extension by CMCM to cover a defined region of proteinsequence space (Reetz et al., Angew. Chem. Int. Ed Engl. 40:3589-3591(2001)). As with CCM, this method can test virtually all possiblealterations over a target region. If used along with methods to createrandom mutations and shuffled genes, it provides an excellent means ofgenerating diverse, shuffled proteins. This approach was successful inincreasing, by 51-fold, the enantioselectivity of an enzyme.

In the Mutator Strains technique, conditional is mutator plasmids allowincreases of 20 to 4000-X in random and natural mutation frequencyduring selection and block accumulation of deleterious mutations whenselection is not required (Selifonova et al., Appl. Environ. Microbiol.67:3645-3649 (2001)). This technology is based on a plasmid-derivedmutD5 gene, which encodes a mutant subunit of DNA polymerase III. Thissubunit binds to endogenous DNA polymerase III and compromises theproofreading ability of polymerase III in any strain that harbors theplasmid. A broad-spectrum of base substitutions and frameshift mutationsoccur. In order for effective use, the mutator plasmid should be removedonce the desired phenotype is achieved; this is accomplished through atemperature sensitive (ts) origin of replication, which allows forplasmid curing at 41° C. It should be noted that mutator strains havebeen explored for quite some time (see Low et al., J. Mol. Biol.260:359-3680 (1996)). In this technique, very high spontaneous mutationrates are observed. The conditional property minimizes non-desiredbackground mutations. This technology could be combined with adaptiveevolution to enhance mutagenesis rates and more rapidly achieve desiredphenotypes.

Look-Through Mutagenesis (LTM) is a multidimensional mutagenesis methodthat assesses and optimizes combinatorial mutations of selected aminoacids (Rajpal et al., Proc. Natl. Acad. Sci. USA 102:8466-8471 (2005)).Rather than saturating each site with all possible amino acid changes, aset of nine is chosen to cover the range of amino acid R-groupchemistry. Fewer changes per site allows multiple sites to be subjectedto this type of mutagenesis. A >800-fold increase in binding affinityfor an antibody from low nanomolar to picomolar has been achievedthrough this method. This is a rational approach to minimize the numberof random combinations and can increase the ability to find improvedtraits by greatly decreasing the numbers of clones to be screened. Thishas been applied to antibody engineering, specifically to increase thebinding affinity and/or reduce dissociation. The technique can becombined with either screens or selections.

Gene Reassembly is a DNA shuffling method that can be applied tomultiple genes at one time or to create a large library of chimeras(multiple mutations) of a single gene (Tunable GeneReassembly™ (TGR™)Technology supplied by Verenium Corporation). Typically this technologyis used in combination with ultra-high-throughput screening to query therepresented sequence space for desired improvements. This techniqueallows multiple gene recombination independent of homology. The exactnumber and position of cross-over events can be pre-determined usingfragments designed via bioinformatic analysis. This technology leads toa very high level of diversity with virtually no parental genereformation and a low level of inactive genes. Combined with GSSM™, alarge range of mutations can be tested for improved activity. The methodallows “blending” and “fine tuning” of DNA shuffling, for example, codonusage can be optimized.

In Silico Protein Design Automation (PDA) is an optimization algorithmthat anchors the structurally defined protein backbone possessing aparticular fold, and searches sequence space for amino acidsubstitutions that can stabilize the fold and overall protein energetics(Hayes et al., Proc. Natl. Acad. Sci. USA 99:15926-15931 (2002)). Thistechnology uses in silico structure-based entropy predictions in orderto search for structural tolerance toward protein amino acid variations.Statistical mechanics is applied to calculate coupling interactions ateach position. Structural tolerance toward amino acid substitution is ameasure of coupling. Ultimately, this technology is designed to yielddesired modifications of protein properties while maintaining theintegrity of structural characteristics. The method computationallyassesses and allows filtering of a very large number of possiblesequence variants (10⁵⁰). The choice of sequence variants to test isrelated to predictions based on the most favorable thermodynamics.Ostensibly only stability or properties that are linked to stability canbe effectively addressed with this technology. The method has beensuccessfully used in some therapeutic proteins, especially inengineering immunoglobulins. In silico predictions avoid testingextraordinarily large numbers of potential variants. Predictions basedon existing three-dimensional structures are more likely to succeed thanpredictions based on hypothetical structures. This technology canreadily predict and allow targeted screening of multiple simultaneousmutations, something not possible with purely experimental technologiesdue to exponential increases in numbers.

Iterative Saturation Mutagenesis (ISM) involves: 1) using knowledge ofstructure/function to choose a likely site for enzyme improvement; 2)performing saturation mutagenesis at chosen site using a mutagenesismethod such as Stratagene QuikChange (Stratagene; San Diego Calif.); 3)screening/selecting for desired properties; and 4) using improvedclone(s), start over at another site and continue repeating until adesired activity is achieved (Reetz et al., Nat. Protoc. 2:891-903(2007); and Reetz et al., Angew. Chem. Int. Ed Engl. 45:7745-7751(2006)). This is a proven methodology, which assures all possiblereplacements at a given position are made for screening/selection.

Any of the aforementioned methods for mutagenesis can be used alone orin any combination. Additionally, any one or combination of the directedevolution methods can be used in conjunction with adaptive evolutiontechniques, as described herein.

EXAMPLE I Exemplary Methacrylic Acid Pathway Enzymes

This example describes exemplary pathways for production of methacrylicacid.

FIG. 1 depicts exemplary pathways to MAA from acetyl-CoA and pyruvatevia intermediate citramalate. Also shown are pathways to MAA fromaconitate. In one pathway acetyl-CoA and pyruvate are first converted tocitramalate by citramalate synthase. Dehydration of citramalate canyield either citraconate (Step B) or mesaconate (Step C). Mesaconate andcitraconate are interconverted by a cis/trans isomerase in Step G.Decarboxylation of mesaconate (Step H) or citraconate (Step C) yieldsMAA. In an alternate pathway, citramalate is formed from acetyl-CoA andpyruvate via a citramalyl-CoA intermediate in Steps D and E, catalyzedby citramalyl-CoA lyase and citramalyl-coA hydrolase, transferase orsynthetase.

Also exemplified are pathways from aconitate to MAA. In one pathway,aconitate is first decarboxylated to itaconate by aconitatedecarboxylase (Step I). Itaconate is then isomerized to citraconate byitaconate delta-isomerase (Step J). Conversion of citraconate to MAAproceeds either directly by decarboxylation or indirectly viamesaconate. In an alternate pathway, the itaconate intermediate is firstconverted to itaconyl-CoA by a CoA transferase or synthetase (Step L).Hydration of itaconyl-CoA yields citramalyl-CoA, which can then beconverted to MAA as described above and shown in FIG. 1.

Table 1 shows enzyme classes that can perform the steps depicted inFIG. 1. Exemplary enzymes are described in further detail below.

TABLE 1 Enzyme classes for the enzymes shown in FIG. 1. Label FunctionStep 2.3.1.a Synthase A 2.8.3.a Coenzyme-A transferase E, L 3.1.2.aThiolester hydrolase (CoA specific) E 4.1.1.a Decarboxylase C, H, J4.1.3.a Lyase D 4.2.1.a Dehydratase B, F, K 5.2.1.a Cis/trans isomeraseG 5.3.3.a Delta-Isomerase I 6.2.1.a Acid-thiol ligase E, L

EC 2.3.1.a Synthase (Step A).

Citramalate synthase (EC 2.3.1.182) catalyzes the conversion ofacetyl-CoA and pyruvate to citramalate and coenzyme A. The enzymeparticipates in the threonine-independent pathway of isoleucinebiosynthesis found in archaea such as Methanocaldococcus jannaschii(Howell et al., J. Bacteriol. 181:331-333 (1999)), Leptospirainterrrogans (Xu et al., J. Bacteriol. 186:5400-5409 (2004)) andGeobacter sulfurreducens (Risso et al., J. Bacteriol. 190:2266-2274(2008)). This enzyme operates in the synthetic direction in vivo, ishighly specific for pyruvate as a substrate, and is typically inhibitedby isoleucine. A recombinant citramalate synthase from M. jannaschiideveloped by directed evolution is highly active and lacks inhibition byisoleucine (Atsumi et al., Appl Environ Microbiol 74:7802-7808 (2008)).Citramalate synthase activity was also demonstrated in the citramalatecycle acetate assimilation pathway of Rhodospirillum rubrum, althoughthe gene associated with this activity was not been identified (Berg etal., Mikrobiologiia 78:22-31 (2009)).

Gene GenBank GI Number Organism CimA Q58787.1 2492795 Methanocaldococcusjannaschii CimA ABK13749.1 116664671 Leptospira interrrogans CimAADI84633.1 298505910 Geobacter sulfurreducens

EC 2.8.3.a CoA Transferase (Step E).

CoA transferases catalyze the reversible transfer of a CoA moiety fromone molecule to another. Two transformations in FIG. 1 utilize a CoAtransferase: conversion of citramalyl-CoA to citramalate (Step E) andactivation of itaconate to itaconyl-CoA (Step L). Citramalyl-CoAtransferase (EC 2.8.3.7 and 2.8.3.11) in Step E of FIG. 1 transfers aCoA moiety from citramalyl-CoA to a donor. A citramalate:succinyl-CoAtransferase enzyme is active in the 3-hydroxypropionate cycle ofglyoxylate assimilation. The enzyme is encoded by sst in Chloroflexusaurantiacus, where it is located upstream of the gene encodingcitramalyl-CoA lyase (Friedmann et al., J. Bacteriol. 188:6460-6468(2006)). This enzyme was cloned, heterologously expressed andcharacterized in E. coli. The enzyme is also active as anitaconate:succinyl-CoA transferase. Similar enzymes are found inRoseiflexus sp. RS-1 and Chloroflexus aggregans by sequence identity andproximity to the citramalyl-CoA lyase gene. A CoA transferasecharacterized in Pseudomonas sp. B2aba exhibits both citramlayl-CoAtransferase and itaconyl-CoA transferase activities, allowing theorganism to grow on both itaconate and citramalate (Cooper et al.,Biochem. J 91:82-91 (1964)). The gene associated with this enzyme hasnot been identified. Citramalyl-CoA transferase activity was alsodetected in cell extracts of Achromobacter xylosoxydans, whichefficiently converts itaconate to citramalate (He et al., J BiosciBioeng. 89:388-391 (2000)). The associated gene is not known, but thegene bears the highest protein sequence similarity Sst.

Gene GenBank GI Number Organism Sst YP_001635864.1 163847820Chloroflexus aurantiacus RoseRS_0050 YP_001274443.1 148654238Roseiflexus sp. RS-1 Cagg_3093 YP_002464387.1 219849954 Chloroflexusaggregans AXYL_05983 YP_003981992.1 311109139 Achromobacter xylosoxydans

Additional candidate enzymes for catalyzing these transformationsinclude the gene products of cat1, cat2, and cat3 of Clostridiumkluyveri, which have been shown to exhibit succinyl-CoA,4-hydroxybutyryl-CoA, and butyryl-CoA transferase activity, respectively(See dorf et al., Proc. Natl. Acad. Sci USA 105:2128-2133 (2008);Sohling et al., J Bacteriol. 178:871-880 (1996)). Similar CoAtransferase activities are also present in Trichomonas vaginalis (vanGrinsven et al., J. Biol. Chem. 283:1411-1418 (2008)) and Trypanosomabrucei (Riviere et al., J. Biol. Chem. 279:45337-45346 (2004)).

Protein GenBank ID GI Number Organism cat1 P38946.1 729048 Clostridiumkluyveri cat2 P38942.2 172046066 Clostridium kluyveri cat3 EDK35586.1146349050 Clostridium kluyveri TVAG_395550 XP_001330176 123975034Trichomonas vaginalis G3 Tb11.02.0290 XP_828352 71754875 Trypanosomabrucei

The glutaconyl-CoA-transferase (EC 2.8.3.12) enzyme from anaerobicbacterium Acidaminococcus fermentans reacts with glutaconyl-CoA and3-butenoyl-CoA (Mack et al., Eur. J. Biochem. 226:41-51 (1994)),substrates similar in structure to 2,3-dehydroadipyl-CoA. The genesencoding this enzyme are gctA and gctB. This enzyme has reduced butdetectable activity with other CoA derivatives including glutaryl-CoA,2-hydroxyglutaryl-CoA, adipyl-CoA, crotonyl-CoA and acrylyl-CoA (Buckelet al., Eur. J. Biochem. 118:315-321 (1981)). The enzyme has been clonedand expressed in E. coli (Mack et al., Eur. J. Biochem. 226:41-51(1994)). Glutaconate CoA-transferase activity has also been detected inClostridium sporosphaeroides and Clostridium symbiosum. Additionalglutaconate CoA-transferase enzymes can be inferred by homology to theAcidaminococcus fermentans protein sequence.

Protein GenBank ID GI Number Organism gctA CAA57199.1 559392Acidaminococcus fermentans gctB CAA57200.1 559393 Acidaminococcusfermentans gctA ACJ24333.1 212292816 Clostridium symbiosum gctBACJ24326.1 212292808 Clostridium symbiosum gctA NP_603109.1 19703547Fusobacterium nucleatum gctB NP_603110.1 19703548 Fusobacteriumnucleatum

A CoA transferase that can utilize acetyl-CoA as the CoA donor isacetoacetyl-CoA transferase, encoded by the E. coli atoA (alpha subunit)and atoD (beta subunit) genes (Korolev et al., Acta Crystallogr. D.Biol. Crystallogr. 58:2116-2121 (2002); Vanderwinkel et al., Biochem.Biophys. Res. Commun. 33:902-908 (1968)). This enzyme has a broadsubstrate range (Sramek et al., Arch. Biochem. Biophys. 171:14-26(1975)) and has been shown to transfer the CoA moiety to acetate from avariety of branched and linear acyl-CoA substrates, includingisobutyrate (Matthies et al., Appl Environ. Microbiol 58:1435-1439(1992)), valerate (Vanderwinkel et al., Biochem. Biophys. Res. Commun.33:902-908 (1968)) and butanoate (Vanderwinkel et al., Biochem. Biophys.Res. Commun. 33:902-908 (1968)). This enzyme is induced at thetranscriptional level by acetoacetate, so modification of regulatorycontrol can be useful for engineering this enzyme into a pathway (Pauliet al., Eur. J Biochem. 29:553-562 (1972)). Similar enzymes exist inCorynebacterium glutamicum ATCC 13032 (Duncan et al., Appl. Environ.Microbiol 68:5186-5190 (2002)), Clostridium acetobutylicum (Cary et al.,Appl. Environ. Microbiol 56:1576-1583 (1990); Wiesenborn et al., Appl.Environ. Microbiol 55:323-329 (1989)), and Clostridiumsaccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem.71:58-68 (2007)).

Gene Accession No. GI Number Organism atoA P76459.1 2492994 Escherichiacoli atoD P76458.1 2492990 Escherichia coli actA YP_226809.1 62391407Corynebacterium glutamicum cg0592 YP_224801.1 62389399 Corynebacteriumglutamicum ctfA NP_149326.1 15004866 Clostridium acetobutylicum ctfBNP_149327.1 15004867 Clostridium acetobutylicum ctfA AAP42564.1 31075384Clostridium saccharoperbutylacetonicum ctfB AAP42565.1 31075385Clostridium saccharoperbutylacetonicum

EC 3.2.1.a CoA Hydrolase (Step E).

Enzymes in the 3.1.2 family hydrolyze acyl-CoA molecules to theircorresponding acids. Several CoA hydrolases with broad substrate rangesare suitable candidates for exhibiting citramalyl-CoA hydrolaseactivity. For example, the enzyme encoded by acotI2 from Rattusnorvegicus brain (Robinson et al., Biochem. Biophys. Res. Commun.71:959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoA andmalonyl-CoA. The human dicarboxylic acid thioesterase, encoded by acot8,exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA, sebacyl-CoA,and dodecanedioyl-CoA (Westin et al., J. Biol. Chem. 280:38125-38132(2005)). The closest E. coli homolog to this enzyme, tesB, can alsohydrolyze a range of CoA thiolesters (Naggert et al., J Biol Chem266:11044-11050 (1991)). A similar enzyme has also been characterized inthe rat liver (Deana R., Biochem Int 26:767-773 (1992)). Additionalenzymes with hydrolase activity in E. coli include ybgC, paaI, and ybdB(Kuznetsova, et al., FEMS Microbiol Rev, 2005, 29(2):263-279; Song etal., J Biol Chem, 2006, 281(16):11028-38). Though its sequence has notbeen reported, the enzyme from the mitochondrion of the pea leaf has abroad substrate specificity, with demonstrated activity on acetyl-CoA,propionyl-CoA, butyryl-CoA, palmitoyl-CoA, oleoyl-CoA, succinyl-CoA, andcrotonyl-CoA (Zeiher et al., Plant. Physiol. 94:20-27 (1990)). Theacetyl-CoA hydrolase, ACH1, from S. cerevisiae represents anothercandidate hydrolase (Buu et al., J. Biol. Chem. 278:17203-17209 (2003)).

Gene name GenBank ID GI Number Organism acot12 NP_570103.1 18543355Rattus norvegicus tesB NP_414986 16128437 Escherichia coli acot8CAA15502 3191970 Homo sapiens acot8 NP_570112 51036669 Rattus norvegicustesA NP_415027 16128478 Escherichia coli ybgC NP_415264 16128711Escherichia coli paaI NP_415914 16129357 Escherichia coli ybdB NP_41512916128580 Escherichia coli ACH1 NP_009538 6319456 Saccharomycescerevisiae

Yet another candidate hydrolase is the glutaconate CoA-transferase fromAcidaminococcus fermentans. This enzyme was transformed by site-directedmutagenesis into an acyl-CoA hydrolase with activity on glutaryl-CoA,acetyl-CoA and 3-butenoyl-CoA (Mack et al., FEBS. Lett. 405:209-212(1997)). This indicates that the enzymes encodingsuccinyl-CoA:3-ketoacid-CoA transferases and acetoacetyl-CoA:acetyl-CoAtransferases can also serve as candidates for this reaction step, withappropriate mutations introduced to change their function as describedabove.

Gene GenBank GI Number Organism gctA CAA57199 559392 Acidaminococcusfermentans gctB CAA57200 559393 Acidaminococcus fermentans

EC 4.1.1.a Decarboxylase (Steps C, H, J).

The final step of MAA in FIG. 1 is the decarboxylation of eithermesaconate or citraconate. Although enzymes with citraconate ormesaconate decarboxylase activity have not been identified to date,suitable enzyme candidates include aconitate decarboxylase (EC 4.1.1.6),which catalyzes the conversion of aconitate to itaconate,4-oxalocrotonate decarboxylase (EC 4.1.1.77), which catalyzes theconversion of 4-oxalocrotonate to 2-oxopentenoate, and enzymes in thecinnamate decarboxylase family (EC 4.1.1.-). Aconitate decarboxylase (EC4.1.1.6) catalyzes the final step in itaconate biosynthesis in a strainof Candida and also in the filamentous fungus Aspergillus terreus(Bonnarme et al. J. Bacteriol. 177:3573-3578 (1995); Willke and Vorlop,Appl Microbiol. Biotechnol 56:289-295 (2001)). A cis-aconitatedecarboxylase (CAD) (EC 4.1.16) has been purified and characterized fromAspergillus terreus (Dwiarti et al., J. Biosci. Bioeng. 94(1): 29-33(2002)). Recently, the gene has been cloned and functionallycharacterized (Kanamasa et al., Appl. Microbiol Biotechnol 80:223-229(2008)) and (WO/2009/014437). Several close homologs of CAD are listedbelow (EP 2017344A1; WO 2009/014437 A1). The gene and protein sequenceof CAD were reported previously (EP 2017344 A1; WO 2009/014437 A1),along with several close homologs listed below.

Gene GenBank GI Number Organism CAD XP_001209273 115385453 Aspergillusterreus XP_001217495 115402837 Aspergillus terreus XP_001209946115386810 Aspergillus terreus BAE66063 83775944 Aspergillus oryzaeXP_001393934 145242722 Aspergillus niger XP_391316 46139251 Gibberellazeae XP_001389415 145230213 Aspergillus niger XP_001383451 126133853Pichia stipitis YP_891060 118473159 Mycobacterium smegmatis NP_96118741408351 Mycobacterium avium subsp. pratuberculosis YP_880968 118466464Mycobacterium avium ZP_01648681 119882410 Salinispora arenicola

4-Oxalocronate decarboxylase catalyzes the decarboxylation of4-oxalocrotonate to 2-oxopentanoate. This enzyme has been isolated fromnumerous organisms and characterized. Genes encoding this enzyme includedmpH and dmpE in Pseudomonas sp. (strain 600) (Shingler et al.,174:711-724 (1992)), xylII and xylIII from Pseudomonas putida (Kato etal., Arch. Microbiol 168:457-463 (1997); Stanley et al., Biochemistry39:3514 (2000); Lian et al., J. Am. Chem. Soc. 116:10403-10411 (1994))and Reut_B5691 and Reut_B5692 from Ralstonia eutropha JMP134 (Hughes etal., 158:79-83 (1984)). The genes encoding the enzyme from Pseudomonassp. (strain 600) have been cloned and expressed in E. coli (Shingler etal., J. Bacteriol. 174:711-724 (1992)). The 4-oxalocrotonatedecarboxylase encoded by xylI in Pseudomonas putida functions in acomplex with vinylpyruvate hydratase. A recombinant form of this enzymedevoid of the hydratase activity and retaining wild type decarboxylaseactivity has been characterized (Stanley et al., Biochem. 39:718-26(2000)). A similar enzyme is found in Ralstonia pickettii (formerlyPseudomonas pickettii) (Kukor et al., J Bacteriol. 173:4587-94 (1991)).

Gene GenBank GI Number Organism dmpH CAA43228.1 45685 Pseudomonas sp.CF600 dmpE CAA43225.1 45682 Pseudomonas sp. CF600 xylII YP_709328.1111116444 Pseudomonas putida xylIII YP_709353.1 111116469 Pseudomonasputida Reut_B5691 YP_299880.1 73539513 Ralstonia eutropha JMP134Reut_B5692 YP_299881.1 73539514 Ralstonia eutropha JMP134 xylI P49155.11351446 Pseudomonas putida tbuI YP_002983475.1 241665116 Ralstoniapickettii

Finally, a class of decarboxylases has been characterized that catalyzethe conversion of cinnamate (phenylacrylate) and substituted cinnamatederivatives to their corresponding styrene derivatives. These enzymesare common in a variety of organisms and specific genes encoding theseenzymes that have been cloned and expressed in E. coli include: pad 1from Saccharomyces cerevisae (Clausen et al., Gene 142:107-112 (1994)),pdc from Lactobacillus plantarum (Barthelmebs et al., 67:1063-1069(2001); Qi et al., Metab Eng 9:268-276 (2007); Rodriguez et al., J.Agric. Food Chem. 56:3068-3072 (2008)), pofK (pad) from Klebsiellaoxytoca (Uchiyama et al., Biosci. Biotechnol. Biochem. 72:116-123(2008); Hashidoko et al., Biosci. Biotech. Biochem. 58:217-218 (1994)),Pedicoccus pentosaceus (Barthelmebs et al., 67:1063-1069 (2001)), andpadC from Bacillus subtilis and Bacillus pumilus (Shingler et al.,174:711-724 (1992)). A ferulic acid decarboxylase from Pseudomonasfluorescens also has been purified and characterized (Huang et al., J.Bacteriol. 176:5912-5918 (1994)). Enzymes in this class are stable anddo not require either exogenous or internally bound co-factors, thusmaking these enzymes ideally suitable for biotransformations(Sariaslani, Annu. Rev. Microbiol. 61:51-69 (2007)).

Gene name GenBankID GI Number Organism pad1 BAG32372.1 188496949Saccharomyces cerevisae pdc AAC45282.1 1762616 Lactobacillus plantarumpofK (pad) BAF65031.1 149941607 Klebsiella oxytoca padC AAC46254.12394282 Bacillus subtilis pad CAC16793.1 11322457 Pedicoccus pentosaceuspad CAC18719.1 11691810 Bacillus pumilus

Another suitable enzyme is sorbic acid decarboxylase which convertssorbic acid to 1,3-pentadiene. Sorbic acid decarboxylation byAspergillus niger requires three genes: padA1, ohbA1, and sdrA(Plumridge et al. Fung. Genet. Bio, 47:683-692 (2010). PadA1 isannotated as a phenylacrylic acid decarboxylase, ohbA1 is a putative4-hydroxybenzoic acid decarboxylase, and sdrA is a sorbic aciddecarboxylase regulator. Additional species have also been shown todecarboxylate sorbic acid including several fungal and yeast species(Kinderlerler and Hatton, Food Addit Contam., 7(5):657-69 (1990); Casaset al., Int J Food Micro., 94(1):93-96 (2004); Pinches and Apps, Int. J.Food Microbiol. 116: 182-185 (2007)). For example, Aspergillus oryzaeand Neosartorya fischeri have been shown to decarboxylate sorbic acidand have close homologs to padA1, ohbA1, and sdrA.

Gene name GenBankID GI Number Organism padA1 XP_001390532.1 145235767Aspergillus niger ohbA1 XP_001390534.1 145235771 Aspergillus niger sdrAXP_001390533.1 145235769 Aspergillus niger padA1 XP_001818651.1169768362 Aspergillus oryzae ohbA1 XP_001818650.1 169768360 Aspergillusoryzae sdrA XP_001818649.1 169768358 Aspergillus oryzae padA1XP_001261423.1 119482790 Neosartorya fischeri ohbA1 XP_001261424.1119482792 Neosartorya fischeri sdrA XP_001261422.1 119482788 INeosartorya fischeri

Each of the decarboxylases listed above represents a possible suitableenzyme for decarboxylating mesaconate or citraconate. If the desiredactivity or productivity of the enzyme is not observed in the desiredconversions, the decarboxylase enzymes can be evolved using knownprotein engineering methods to achieve the required performance.Importantly, it was shown through the use of chimeric enzymes that theC-terminal region of decarboxylases appears to be responsible forsubstrate specificity (Barthelmebs et al., AEM 67, 1063-1069 (2001)).Accordingly, directed evolution experiments to broaden the specificityof decarboxylases in order to gain activity with mesaconate orcitraconate can be focused on the C-terminal region of these enzymes.

EC 4.1.3.a Lyase (Step D).

Citramalyl-CoA lyase (EC 4.1.3.25) converts acetyl-CoA and pyruvate tocitramalyl-CoA, shown in Step D of FIG. 1. This enzyme participates inthe 3-hydroxypropionate (3-HP) cycle of glyoxylate assimilation, whereit acts in the citramalyl-CoA degrading direction. The enzyme is encodedby the ccl gene in the green nonsulfur phototrophic bacteriumChloroflexus aurantiacus (Friedmann et al., J. Bacteriol. 189:2906-2914(2007)), where it is located downstream of a gene encodingcitramalyl-CoA transferase. The ccl gene was cloned and heterologouslyexpressed in E. coli. Similar gene clusters are found in Roseiflexus sp.RS-1 and Chloroflexus aggregans, although the enzymes have not beencharacterized to date. The 3-HP cycle is also active in Rhodobactersphaeroides, whose genome encodes a protein with high sequencesimilarity to the ccl gene product (Filatova et al., Mikrobiologiia74:319-328 (2005)). The citramalyl-CoA lyase from a Bacillus sp. wasshown to be reversible, although the associated gene has not beenidentified to date (Sasaki et al., J. Biochem. 73:599-608 (1973)).

Gene GenBank GI Number Organism Caur_2265 YP_001635863.1 163847819Chloroflexus aurantiacus RoseRS_0049 YP_001274442.1 148654237Roseiflexus sp. RS-1 Cagg_3093 YP_002464386.1 219849953 Chloroflexusaggregans Rsph17029_1172 YP_001043054.1 126461940 Rhodobactersphaeroides

EC 4.2.1.a Dehydratase (Steps B, F).

The dehydration of citramalate to citraconate in Step B of FIG. 1 iscatalyzed by an enzyme with citramalate dehydratase (citraconateforming) activity (EC 4.2.1.35). This enzyme, along with citramalatesynthase, participates in the threonine-independent isoleucinebiosynthesis pathway characterized in Methanocaldococcus jannaschii andLeptospira interrrogans. The dehydration of citramalate in theseorganisms catalyzed by isopropylmalate isomerase (IPMI), which catalyzesboth the dehydration of citramalate to citraconate and the subsequenttrans-addition of water to citraconate to form methylmalate (Xu et al.,J. Bacteriol. 186:5400-5409 (2004); Drevland et al., J. Bacteriol.189:4391-4400 (2007)). The M. jannaschii homoaconitase (EC 4.2.1.114)encoded by hacAB is also a suitable candidate, and mutants of thisenzyme with altered substrate specificity and isopropylmalate isomeraseactivity have been characterized (Jeyakanthan et al., Biochemistry49:2687-2696 (2010)). The L. interrogans IPMI genes were cloned into E.coli, where they were able to complement strains deficient in nativeaconitase activity when expressed together (Xu et al., J. Bacteriol.186:5400-5409 (2004)). Citramalate dehydratase has also beencharacterized in Pseudomonas putida, where it participates in3,5-xylenol degradation. The genes encoding the enzymes of this pathway,including citramalate dehydratase, are located on the transmissibleplasmid pRA500, and they have been cloned into E. coli (Jain, Appl.Microbiol. Biotechnol., 45:502-508 (1996)). Citraconate is also asubstrate of the Saccharomyces cerevisiae 3-isopropylmalate dehydratase(EC 4.2.1.33) enzyme encoded by LEU1 (Kohlhaw, Methods Enzymol.166:423-429 (1988)). The 3-isopropylmalate dehydratase LEU1 of Candidamaltosa exhibits higher activity on citraconate than the nativesubstrate (Bode, R. and Birnbaum, D., J. Basic Microbiol. 31:21-26(1991)). Attenuation or selective inhibition of the citraconate tomethylmalate hydration activity may be required to increase accumulationof the citraconate intermediate.

Gene GenBank GI Number Organism LeuC P81291.1 3219823 Methanocaldococcusjannaschii LeuD Q58673.1 3122345 Methanocaldococcus jannaschii hacAQ58409.1 3122347 Methanocaldococcus jannaschii hacB Q58667.1 3122344Methanocaldococcus jannaschii LeuC NP_712276.1 24214795 Leptospirainterrrogans LeuD NP_712277.1 24214796 Leptospira interrrogans LEU1AAB19612.1 234318 Saccharomyces cerevisiae cmLEU1 AAB03335.1 1399939Candida maltosa

Another suitable citramalate dehydratase candidate is the2-methylcitrate dehydratase enzymes that form cis-2-methylaconitate (EC4.2.1.79) and trans-2-methylaconitate (EC 4.2.1.117). The2-methylcitrate cis-dehydratase of E. coli encoded by prpD is active oncitramalate as a substrate (Blank et al., Microbiology 148:133-146(2002)). Neither citraconate nor mesaconate were active as substrates,indicating that PrpD is strictly a dehydratase enzyme rather than anisomerase. The PrpD enzyme of Salmonella enterica serovar Typhimuriumhas also been characterized, but activity on citramalate has not beendemonstrated (Horswill et al., Biochemistry 40:4703-4713 (2001)). The2-methylcitrate dehydratase enzymes of Shewanella oneidensis encoded byacnB and acnD are also candidates (Grimek et al., J. Bacteriol.186:454-462 (2004)). The AcnD enzyme requires a cofactor encoded by prpFto function in vivo. Crystal structure studies of PrpF in complex withaconitate show that the enzyme functions as a cis/trans isomerase,interconverting the cis/trans isomers of aconitate and 2-methylaconitate(Garvey et al., Protein Sci 16:1274-1284 (2007)). An additionalcandidate from S. oneidensis is a predicted isopropylmalate isomeraseencoded by leuCD.

Gene GenBank GI Number Organism prpD AAC73437.1 1786528 Escherichia coliprpD AAC44816.1 1648968 Salmonella enterica serovar Typhimurium acnBNP_716069.1 24372027 Shewanella oneidensis acnD AAN53428.1 24345782Shewanella oneidensis prpF AAN53427.1 24345781 Shewanella oneidensisleuC NP_719761.1 24375718 Shewanella oneidensis leuD NP_719760.124375717 Shewanella oneidensis

The dehydration of citramalate to mesaconate in Step F is catalyzed bycitramalate dehydratase (mesaconate forming, EC 4.2.1.34), also called2-methylmalate dehydratase or mesaconase. This enzyme has been onlypartially characterized in Clostridium tetanomorphum, and genecandidates are not available to date. The activity has also beendemonstrated in cell extracts of Rhodospirillum rubrum, where itparticipates in the citramalate cycle of acetate utilization (Berg andIvanovskii, Mikrobiologiia 78:22-31 (2009)), although the associatedgene has not been identified.

Itaconyl-CoA is converted to citramalyl-CoA (Step K of FIG. 1) byitaconyl-CoA hydratase (EC 4.2.1.56), an enzyme that participates initaconate assimilation pathways in organisms such as Pseudomonas sp.B2aba (Cooper and Kornberg, Biochem. J 91:82-91 (1964)), Achromobacterxylosoxydans (He et al., J Biosci Bioeng. 89:388-391 (2000)) andPseudomonas fluorescens (Nagai, J., J. Biochem, 53:181-7 (1963)). Thisenzyme activity has not been associated with a gene to date.

EC 5.2.1.a Cis/Trans Isomerase (Step G).

The cis/trans isomerization of mesaconate and citraconate is catalyzedby an enzyme with citraconate isomerase activity. Suitable candidatesinclude aconitate isomerase (EC 5.3.3.7), maleate cis, trans-isomerase(EC 5.2.1.1), maleylacetone cis, trans-isomerase and cis,trans-isomerase of unsaturated fatty acids (Cti).

Aconitate isomerase interconverts cis- and trans-aconitate. Theaconitate isomerase of Shewanella oneidensis, encoded by prpF,interconverts the cis/trans isomers of aconitate and 2-methylaconitate(Garvey et al., Protein Sci 16:1274-1284 (2007)). This enzyme operatesin complex with the methylcitrate dehydratase, AcnD. Aconitate isomeraseactivity was detected in many gram-negative bacteria includingPseudomonas fluorescens and Pseudomonas putida, but not in gram-positivebacteria (Watanabe et al., Curr Microbiol 35:97-102 (1997)). Purifiedenzyme from Pseudomonas putida has been characterized (Klinman et al.,Biochemistry 10:2253-2259 (1971)). Aconitate isomerase enzymes have alsobeen studied in plants, but the genes have not been identified to date(Thompson et al., Anal. Biochem. 184:39-47 (1990)). Some predictedproteins with high sequence homology to the prpF protein are listedbelow.

Gene GenBank GI Number Organism prpF AAN53427.1 24345781 Shewanellaoneidensis prpF PP_2337 26989061 Pseudomonas putida Pfl01_1767YP_347499.1 77457994 Pseudomonas fluorescens Reut_A1811 YP_296020.173541500 Ralstonia eutropha

Therefore, the addition of a cis, trans isomerase may help to improvethe yield of terepthalic acid. Enzymes for similar isomeric conversionsinclude maleate cis, trans-isomerase (EC 5.2.1.1), maleylacetonecis-trans-isomerase (EC 5.2.1.2), and cis, trans-isomerase ofunsaturated fatty acids (Cti).

Maleate cis, trans-isomerase (EC 5.2.1.1) catalyzes the conversion ofmaleic acid in cis formation to fumarate in trans formation (Scher etal., J Biol. Chem. 244:1878-1882 (1969)). The Alcalidgenes faecalis maiAgene product has been cloned and characterized (Hatakeyama et al.,Biochem. Biophys. Res. Commun. 239:74-79 (1997)). Other maleate cis,trans-isomerases are available in Serratia marcescens (Hatakeyama etal., Biosci. Biotechnol Biochem. 64:1477-1485 (2000)), Ralstoniaeutropha and Geobacillus stearothermophilus.

Gene GenBank GI Number Organism maiA BAA23002 2575787 Alcaligenesfaecalis maiA YP_725437 113866948 Ralstonia eutropha H16 maiA BAA772964760466 Geobacillus stearothermophilus maiA BAA96747.1 8570038 Serratiamarcescens

Maleylacetone cis, trans-isomerase (EC 5.2.1.2) catalyzes the conversionof 4-maleyl-acetoacetate to 4-fumaryl-acetyacetate, a cis to transconversion. This enzyme is encoded by maiA in Pseudomonas aeruginosa(Fernandez-Canon et al., J. Biol. Chem. 273:329-337 (1998)) and Vibriocholera (Seltzer, J. Biol. Chem. 248:215-222 (1973)). A similar enzymewas identified by sequence homology in E. coli O157.

Gene GenBank GI Number Organism maiA NP_250697 15597203 Pseudomonasaeruginosa maiA NP_230991 15641359 Vibrio cholerae maiA EDU73766189355347 Escherichia coli O157

The cti gene product catalyzes the conversion of cis-unsaturated fattyacids (UFA) to trans-UFA. The enzyme has been characterized in P. putida(Junker et al., J Bacteriol. 181:5693-5700 (1999)). Similar enzymes arefound in Shewanella sp. MR-4 and Vibrio cholerae.

Gene GenBank GI Number Organism cti AAD41252 5257178 Pseudomonas putidacti YP_732637 113968844 Shewanella sp. MR-4 cti ZP_04395785 229506276Vibrio cholerae

EC 5.3.3.a Delta-Isomerase (Steps I).

The conversion of itaconate to citraconate is catalyzed by itaconatedelta-isomerase. An enzyme with this activity is the methylitaconatedelta-isomerase (EC 5.3.3.6) of Eubacterium barkeri (Velarde et al., JMol Biol 391:609-620 (2009)). This enzyme was heterologously expressedand characterized in E. coli. Homologs with high protein sequencesimilarity are listed below.

Gene GenBank GI Number Organism mii Q0QLE6.1 122953534 Eubacteriumbarkeri FMAG_01516 ZP_04567526.1 237737045 Fusobacterium mortiferumBACCAP_02290 ZP_02036679.1 154498301 Bacteroides capillosus CLJU_c30450YP_003781195.1 300856211 Clostridium ljungdahlii BMD_3790 YP_003598973.1295705898 Bacillus megaterium EFER_3666 YP_002384731.1 218550940Escherichia fergusonii

EC 6.2.1 CoA Synthetase (Step E).

The conversion of citramalyl-CoA to citramalate (FIG. 1, Step E) anditaconate to itaconyl-CoA (FIG. 1, Step L) can be catalyzed by a CoAacid-thiol ligase or CoA synthetase in the 6.2.1 family of enzymes. Asuccinyl-CoA synthetase enzyme with itaconyl-CoA synthetase (EC 6.2.1.4)activity was characterized in Pseudomonas sp. B2aba, but the gene hasnot been identified (Cooper and Kornberg, Biochem. J 91:82-91 (1964)).Additional CoA ligase enzyme candidates include succinyl-CoA synthetase(EC 6.3.1.4), acetyl-CoA synthetase (EC 6.2.1.13), acyl-CoA ligase andmalonyl-CoA synthetase (EC 6.3.4.9). Several enzymes with broadsubstrate ranges have been described in the literature. ADP-formingacetyl-CoA synthetase (ACD, EC 6.2.1.13) is an enzyme that couples theconversion of acyl-CoA esters to their corresponding acids with theconcomitant synthesis of ATP. ACD I from Archaeoglobus fulgidus, encodedby AF 1211, was shown to operate on a variety of linear andbranched-chain substrates including isobutyrate, isopentanoate, andfumarate (Musfeldt et al., J. Bacteriol. 184:636-644 (2002)). A secondreversible ACD in Archaeoglobus fulgidus, encoded by AF 1983, was alsoshown to have a broad substrate range with high activity on cycliccompounds phenylacetate and indoleacetate (Musfeldt and Schonheit, J.Bacteriol. 184:636-644 (2002)). The enzyme from Haloarcula marismortui(annotated as a succinyl-CoA synthetase) accepts propionate, butyrate,and branched-chain acids (isovalerate and isobutyrate) as substrates,and was shown to operate in the forward and reverse directions (Brasenet al., Arch. Microbiol 182:277-287 (2004)). The ACD encoded by PAE3250from hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed thebroadest substrate range of all characterized ACDs, reacting withacetyl-CoA, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA(Brasen and Schonheit, Arch. Microbiol 182:277-287 (2004)). Directedevolution or engineering can be used to modify this enzyme to operate atthe physiological temperature of the host organism. The enzymes from A.fulgidus, H. marismortui and P. aerophilum have all been cloned,functionally expressed, and characterized in E. coli (Brasen andSchonheit, Arch. Microbiol 182:277-287 (2004); Musfeldt and Schonheit,J. Bacteriol. 184:636-644 (2002)). The acyl-CoA ligase from Pseudomonasputida encoded by paaF has been demonstrated to work on severalaliphatic substrates including acetic, propionic, butyric, valeric,hexanoic, heptanoic, and octanoic acids and on aromatic compounds suchas phenylacetic and phenoxyacetic acids (Fernandez-Valverde et al.,Appl. Environ. Microbiol. 59:1149-1154 (1993)). A related enzyme,malonyl CoA synthetase (6.3.4.9) from Rhizobium leguminosarum canconvert several diacids, namely, ethyl-, propyl-, allyl-, isopropyl-,dimethyl-, cyclopropyl-, cyclopropylmethylene-, cyclobutyl-, andbenzyl-malonate into their corresponding monothioesters (Pohl et al., J.Am. Chem. Soc. 123:5822-5823 (2001)). Additional candidates are thesuccinyl-CoA synthetase enzymes encoded by sucCD in E. coli and LSC12 inS. cerevisiae, which naturally catalyze the formation of succinyl-CoAfrom succinate with the concomitant consumption of one ATP, a reactionwhich is reversible in vivo (Buck et al., Biochemistry 24:6245-6252(1985)).

Protein GenBank ID GI Number Organism AF1211 NP_070039.1 11498810Archaeoglobus fulgidus AF1983 NP_070807.1 11499565 Archaeoglobusfulgidus scs YP_135572.1 55377722 Haloarcula marismortui PAE3250NP_560604.1 18313937 Pyrobaculum aerophilum str. IM2 paaF AAC24333.222711873 Pseudomonas putida matB AAC83455.1 3982573 Rhizobiumleguminosarum sucC NP_415256.1 16128703 Escherichia coli sucD AAC73823.11786949 Escherichia coli LSC1 NP_014785 6324716 Saccharomyces cerevisiaeLSC2 NP_011760 6321683 Saccharomyces cerevisiae

EXAMPLE II Preparation of a MAA Producing Microbial Organism Having anActyl-CoA to MAA Pathway

This example describes the generation of a microbial organism capable ofproducing MAA from acetyl-CoA and pyruvate. This exemplary pathway isshown in Steps A/B/C of FIG. 1.

Escherichia coli is used as a target organism to engineer aMAA-producing pathway from acetyl-CoA and pyruvate as shown in FIG. 1 insteps A, B and C. E. coli provides a good host for generating anon-naturally occurring microorganism capable of producing MAA. E. coliis amenable to genetic manipulation and is known to be capable ofproducing various products, like ethanol, acetic acid, formic acid,lactic acid, succinic acid and 1,4-butanediol, effectively underanaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce MAA from acetyl-CoA,nucleic acids encoding the enzymes utilized in the pathway of FIG. 1,described previously, are expressed in E. coli using well knownmolecular biology techniques (see, for example, Sambrook et al,Molecular Cloning: A Laboratory Manual, Third Ed. (2001); Ausubel et al,Current Protocols in Molecular Biology (1999)). In particular, an E.coli strain is engineered to produce MAA from acetyl-CoA via the routeoutlined in FIG. 1 (Steps A/B/C). Genes encoding enzymes to transformacetyl-CoA to MAA are assembled onto vectors. In particular, the genescimA (Q58787.1), leuCD (P81291.1 and Q58673.1) and cad(XP_(—)001209273), encoding citramalate synthase, citramalatedehydratase and citraconate decarboxylase, respectively, are cloned intothe pZE13 vector (Expressys, Ruelzheim, Germany), under the control ofthe PA1/lacO promoter. The plasmid is then transformed into the hoststrain E. coli MG1655 containing lacI^(Q), which allows inducibleexpression by addition of isopropyl-beta-D-1-thiogalactopyranoside(IPTG). The resulting strain expresses the proteins and enzymes requiredfor synthesis of MAA from acetyl-CoA.

The resulting genetically engineered organism is cultured in glucosecontaining medium following procedures well known in the art (see, forexample, Sambrook et al., supra, 2001). The expression of MAA pathwaygenes is corroborated using methods well known in the art fordetermining polypeptide expression or enzymatic activity, including forexample, Northern blots, PCR amplification of mRNA and immunoblotting.Enzymatic activities of the expressed enzymes are confirmed using assaysspecific for the individually activities. The ability of the engineeredE. coli strain to produce MAA is confirmed using HPLC, gaschromatography-mass spectrometry (GCMS) or liquid chromatography-massspectrometry (LCMS).

Microbial strains engineered to have a functional MAA synthesis pathwayare further augmented by optimization for efficient utilization of thepathway. Briefly, the engineered strain is assessed to determine whetherany of the exogenous genes are expressed at a rate limiting level.Expression is increased for any enzymes expressed at low levels that canlimit the flux through the pathway by, for example, introduction ofadditional gene copy numbers or codon optimization. Strategies are alsoapplied to alter activity, regulation and/or expression of MAA pathwayenzymes, such as mutagenesis and/or directed evolution.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design optional geneknockouts that additionally optimize utilization of the pathway (see,for example, U.S. patent publications US 2002/0012939, US 2003/0224363,US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 andUS 2004/0009466, and in U.S. Pat. No. 7,127,379). Modeling analysisallows reliable predictions of the effects on cell growth of shiftingthe metabolism towards more efficient production of MAA. One modelingmethod is the bilevel optimization approach, OptKnock (Burgard et al.,Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to selectgene knockouts that collectively result in better production of MAA.Adaptive evolution also can be used to generate better producers of, forexample, the citraconate intermediate or the MAA product. Adaptiveevolution is performed to improve tolerance, growth and productioncharacteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004);Alper et al., Science 314:1565-1568 (2006)). Based on the results,subsequent rounds of modeling, genetic engineering and adaptiveevolution can be applied to the MAA producer to further increaseproduction.

For large-scale production of MAA, the above MAA pathway-containingorganism is cultured in a fermenter using a medium known in the art tosupport growth of the organism under anaerobic conditions. Fermentationsare performed in either a batch, fed-batch or continuous manner.Anaerobic conditions are maintained by first sparging the medium withnitrogen and then sealing culture vessel (e.g., flasks can be sealedwith a septum and crimp-cap). Microaerobic conditions also can beutilized by providing a small hole for limited aeration. The pH of themedium is maintained at a pH of 7 by addition of an acid, such as H₂SO₄.The growth rate is determined by measuring optical density using aspectrophotometer (600 nm), and the glucose uptake rate by monitoringcarbon source depletion over time. Byproducts such as undesirablealcohols, organic acids, and residual glucose can be quantified by HPLC(Shimadzu) with an HPX-087 column (BioRad), using a refractive indexdetector for glucose and alcohols, and a UV detector for organic acids,Lin et al., Biotechnol. Bioeng., 775-779 (2005).

EXAMPLE III Exemplary Enzymes for the Formation of Methacrylate Estersfrom Methacrylate and Alcohols

This example describes enzymes for an exemplary pathway for productionof methacrylate esters.

FIG. 2 depicts an exemplary pathway from MAA and alcohols tomethacrylate esters. A methacrylyl-CoA synthetase or methacrylyl-CoAtransferase can be applied to form methacrylyl-CoA from methacrylate.Enzymes with alcohol transferase activity can be applied to formmethacrylate esters from methacrylyl-CoA and alcohols.

The conversion of methacrylate to methacrylyl-CoA can be catalyzed by aCoA acid-thiol ligase or CoA synthetase in the 6.2.1 family of enzymes.Exemplary enzymes with this activity are provided in Example I.Alternatively, conversion of methacrylate to methacrylyl-CoA can becatalyzed by a transferase enzyme in the 2.8.3 family of enzymes. CoAtransferases catalyze the transfer of a CoA moiety from one molecule toanother. Exemplary enzymes with this activity are provided in Example I.

Formation of methacrylate esters from methacrylyl-CoA and alcohols iscatalyzed by enzymes having alcohol transferase activity. Severalenzymes with alcohol transferase activity were demonstrated in Examples1-10 of U.S. Pat. No. 7,901,915. These include Novozyme 435 (immobilizedlipase B from Candida antarctica, Sigma), Lipase C2 from Candidacylindracea (Alphamerix Ltd), lipase from Pseudomonas fluorescens(Alphamerix Ltd), L-aminoacylase ex Aspergillus spp., and protease exAspergillus oryzae. Such enzymes were shown to form methyl acrylate andethyl acrylate from acrylyl-CoA and methanol or ethanol, respectively.Such transferase enzymes can therefore be used to form methacrylateesters.

Other suitable enzymes include the lipase encoded by calB from Candidaantarctica (Efe et al., Biotechnol. Bioeng. 99:1392-1406 (2008)) and theEstF1 esterase from Pseudomonas fluorescens (Khalameyzer et al., Appl.Environ. Microbiol. 65:477-482 (1999)). Lipase enzymes encoded by lipBfrom Pseudomonas fluorescens and estA from Bacillus subtilis may alsocatalyze this transformation. The B. subtilis and P. fluorescens genesencode triacylglycerol lipase enzymes which have been cloned andcharacterized in E. coli (Dartois et al., Biochim. Biophys. Acta1131:253-260 (1992); Tan et al., Appl. Environ. Microbiol. 58:1402-1407(1992)).

Gene Accession No. GI No. Organism calB P41365.1 1170790 Candidaantarctica EstF1 AAC36352.1 3641341 Pseudomonas fluorescens lipBP41773.1 1170792 Pseudomonas fluorescens estA P37957.1 7676155 Bacillussubtilis

Additional candidate genes that encode enzymes for forming methacrylateesters from methacrylyl-CoA and alcohols include the Acinetobacter sp.ADP1 atfA encoding a bifunctional enzyme with both wax ester synthase(WS) and acyl-CoA:diacylglycerol acyltransferase (DGAT) activities(Kalscheuer et al. AJ Biol Chem 2003, 278: 8075-8082); the Simmondsiachinensis gene AAD38041 encoding a enzyme required for the accumulationof waxes in jojoba seeds (Lardizabal et al. Plant Physiology 2000, 122:645-655); the Alcanivorax borkumensis atfA1 and atfA2 encodingbifunctional WS/DGAT enzymes (Kalscheuer et al. J Bacteriol 2007, 189:918-928); ths Fragaria×ananassa AAT encoding an alcoholacetyltransferasae (Noichinda et al. Food Sci Technol Res 1999, 5:239-242); the Rosa hybrid cultivar AAT1 encoding an alcoholacetyltransferase (Guterman et al. Plant MoI Biol 2006, 60: 555-563);the Saccharomyces cerevisiae ATF1 and ATF2 encoding alcoholacetyltransferases (Mason et al. Yeast 2000, 16: 1287-1298); and Ws1 andWs2 from Marinobacter hydrocarbonoclasticus (Holtzapple, E. andSchmidt-Dannert, C., J. Bacteriol. 189 (10), 3804-3812, 2007). Thecarboxylesterase from Lactococcus lactis, encoded by estA, catalyzes theformation of esters from acetyl-CoA and alcohols such as ethanol andmethanethiol (Nardi et al. J. Appl. Microbiol. 93:994-1002 (2002)). Thealcohol O-acetyltransferase from Saccharomyces uvarum converts a widerange of alcohol substrates including branched-chain alcohols to theircorresponding acetate esters (Yoshioka and Hashimoto, Agricul and BiolChem, 45:2183-2191 (1981). The protein sequences of the enzymes encodedby these genes are provided below.

GI Gene GenBank ID Number Organism atfA Q8GGG1 81478805 Acinetobactersp. ADP1 AF149919.1: AAD38041 5020219 Simmondsia chinensis 13 . . . 1071atfAl YP694462 110835603 Alcanivorax borkumensis SK2 atfA2 YP693524110834665 Alcanivorax borkumensis SK2 AAT AAG13130.1 10121328 Fragaria ×ananassa AATl Q5I6B5 75105208 Rosa hybrid cultivar ATFl P40353 2506980Saccharomyces cerevisiae ATF2 P53296 1723729 Saccharomyces cerevisiaeWs2 ABO21021.1 126567232 Marinobacter hydrocarbonoclasticus Ws1ABO21020.1 126567230 Marinobacter hydrocarbonoclasticus EstA AAF62859.17453516 Lactococcus lactis

EXAMPLE IV Exemplary Enzymes for the Formation of Methacrylate Estersfrom Methacrylate and Alcohols

This example describes enzymes for an exemplary pathway for productionof methacrylate esters.

FIG. 2 depicts an exemplary pathway from MAA and alcohols tomethacrylate esters. Methacrylate esters can be produced chemically, forexample, by heating methacrylate in the presence of an alcohol ormultiple alcohols in the presence of a dehydrating agent such as an acidcatalyst. Enzymes with methacrylate ester-forming activity can also beapplied to form methacrylate esters directly from methacrylate andalcohols. Genes encoding such enzymes can be expressed in an organismcontaining a methacrylate synthesis pathway. Several such organismscontaining methacrylate synthesis pathways are disclosed herein and inWO/2009/135074. The methacrylate ester-forming enzymes can be targetedto the cytosol to enable intracellular conversion of alcohols andmethacrylate to methacrylate esters. Alternatively, the methacrylateester-forming enzymes can be secreted into the fermentation medium toenable extracellular conversion of alcohols and methacrylate tomethacrylate esters. Another option is to add methacrylate ester-formingenzymes to a mixture containing methacrylate and alcohols, such as afermentation broth. Several enzymes with methacrylate-ester formingactivity are described below.

The amidase from Brevibacterium sp. R312 (EC 3.5.1.4) is a likely enzymewith methacrylate ester-forming activity. This enzyme was shown tohydrolyze ethylacrylate (Thiery et al., J. Gen. Microbiol., 132:2205-8,1986; Soubrier et al., Gene, 116:99-104, 1992). The microsomal epoxidehydrolase from Rattus norvegicus (EC 3.3.2.9) has activity onhydrolyzing glycidyl methacrylate and is another suitable enzyme(Guengerich et al., Rev. Biochem. Toxicol. 4:5-30, 1982). The proteinsequences of these genes are provided below.

Gene GenBank ID GI Number Organism amiE JC1174 98711 Brevibacterium sp.Eph-1 P07687.1 123928 Rattus norvegicus

Additional genes encoding potential methacrylate ester-forming enzymesinclude the Acinetobacter sp. ADP1 atfA encoding a bifunctional enzymewith both wax ester synthase (WS) and acyl-CoA:diacylglycerolacyltransferase (DGAT) activities (Kalscheuer et al. AJ Biol Chem 2003,278: 8075-8082); the Simmondsia chinensis gene AAD38041 encoding aenzyme required for the accumulation of waxes in jojoba seeds(Lardizabal et al. Plant Physiology 2000, 122: 645-655); the Alcanivoraxborkumensis atfA1 and atfA2 encoding bifunctional WS/DGAT enzymes(Kalscheuer et al. J Bacteriol 2007, 189: 918-928); theFragaria×ananassa AAT encoding an alcohol acetyltransferasae (Noichindaet al. Food Sci Technol Res 1999, 5: 239-242); the Rosa hybrid cultivarAAT1 encoding an alcohol acetyltransferase (Guterman et al. Plant MoIBiol 2006, 60: 555-563); and the Saccharomyces cerevisiae ATF1 and ATF2encoding alcohol acetyltransferases (Mason et al. Yeast 2000, 16:1287-1298); and Ws1 and Ws2 from Marinobacter hydrocarbonoclasticus(Holtzapple, E. and Schmidt-Dannert, C., J. Bacteriol. 189 (10),3804-3812, 2007). The carboxylesterase from Lactococcus lactis, encodedby estA, catalyzes the formation of esters from acetyl-CoA and alcoholssuch as ethanol and methanethiol (Nardi et al. J. Appl. Microbiol.93:994-1002 (2002)). The alcohol O-acetyltransferase from Saccharomycesuvarum converts a wide range of alcohol substrates includingbranched-chain alcohols to their corresponding acetate esters (Yoshiokaand Hashimoto, Agricul and Biol Chem, 45:2183-2191 (1981). The geneassociated with this activity has not been identified to date. Theprotein sequences of the enzymes encoded by these genes are providedbelow.

Gene GenBank ID GI Number Organism atfA Q8GGG1 81478805 Acinetobactersp. ADP1 AF149919.1: AAD38041 5020219 Simmondsia chinensis 13 . . . 1071atfAl YP694462 110835603 Alcanivorax borkumensis SK2 atfA2 YP693524110834665 Alcanivorax borkumensis SK2 AAT AAG13130.1 10121328 Fragaria ×ananassa AATl Q5I6B5 75105208 Rosa hybrid cultivar ATFl P40353 2506980Saccharomyces cerevisiae ATF2 P53296 1723729 Saccharomyces cerevisiaeWs2 ABO21021.1 126567232 Marinobacter hydrocarbonoclasticus Ws1ABO21020.1 126567230 Marinobacter hydrocarbonoclasticus EstA AAF62859.17453516 Lactococcus lactis

The Homo sapiens paraoxonase enzymes PON1, PON1 (G3C9), and PON3 (EC3.1.8.1) possess both arylesterase and organophosphatase activities andalso may possess methacrylate ester-forming activity. PON1 has a commonpolymorphic site at residue 192, glutamine (R) or arginine (Q), thatresults in qualitative differences. For example, the R isozyme has ahigher esterase activity on GBL than the S isozyme (Billecke et al.,Drug Metab Dispos. 28:1335-1342 (2000)). In H. sapiens cells, PON1resides on high-density lipoprotein (HDL) particles, and its activityand stability require this environment. Wild type and recombinant PON1enzymes have been functionally expressed in other organisms (Rochu etal., Biochem. Soc. Trans. 35:1616-1620 (2007); Martin et al., Appl.Environ. Microbiol. (2009)). A directed evolution study of PON1 yieldedseveral mutant enzymes with improved solubility and catalytic propertiesin E. coli (nucleotide accession numbers AY499188-AY499199) (Aharoni etal., Proc. Natl. Acad. Sci. U.S.A 101:482-487 (2004)). One recombinantvariant from this study, G3C9 (Aharoni et al., Proc. Natl. Acad. Sci.U.S.A 101:482-487 (2004)), was recently used in an integrated bioprocessfor the pH-dependent production of 4-valerolactone from levulinate(Martin et al., Appl. Environ. Microbiol. (2009)). Human PON3 is yetanother suitable enzyme that may possess methacrylate ester-formingactivity (Draganov et al., J. Lipid Res. 46:1239-1247 (2005)).

Gene Accession No. GI No. Organism PON1 NP_000437.3 19923106 Homosapiens PON1 (G3C9) AAR95986.1 40850544 Synthetic variant PON3NP_000931.1 29788996 Homo sapiens

Additionally, the Candida antarctica lipase B is another suitablecandidate enzyme with methacrylate ester-forming activity (Efe et al.,Biotechnol. Bioeng. 99:1392-1406 (2008)). The esterase from Pseudomonasfluorescens, encoded by EstF1, is yet another suitable enzyme(Khalameyzer et al., Appl. Environ. Microbiol. 65:477-482 (1999)). Otherlipase enzymes from organisms such as Pseudomonas fluorescens andBacillus subtilis may also catalyze this transformation. The B. subtilisand P. fluorescens genes encode triacylglycerol lipase enzymes whichhave been cloned and characterized in E. coli (Dartois et al., Biochim.Biophys. Acta 1131:253-260 (1992); Tan et al., Appl. Environ. Microbiol.58:1402-1407 (1992)).

Gene Accession No. GI No. Organism calB P41365.1 1170790 Candidaantarctica EstF1 AAC36352.1 3641341 Pseudomonas fluorescens lipBP41773.1 1170792 Pseudomonas fluorescens estA P37957.1 7676155 Bacillussubtilis

Formation of methacrylate esters may also be catalyzed by enzymes in the3.1.1 family that act on carboxylic ester bonds molecules for theinterconversion between cyclic lactones and the open chainhydroxycarboxylic acids. The L-lactonase from Fusarium proliferatumECU2002 exhibits lactonase and esterase activities on a variety oflactone substrates (Zhang et al., Appl. Microbiol. Biotechnol.75:1087-1094 (2007)). The 1,4-lactone hydroxyacylhydrolase (EC3.1.1.25), also known as 1,4-lactonase or gamma-lactonase, is specificfor 1,4-lactones with 4-8 carbon atoms. The gamma lactonase in humanblood and rat liver microsomes was purified (Fishbein et al., J BiolChem 241:4835-4841 (1966)) and the lactonase activity was activated andstabilized by calcium ions (Fishbein et al., J Biol Chem 241:4842-4847(1966)). The optimal lactonase activities were observed at pH 6.0,whereas high pH resulted in hydrolytic activities (Fishbein and Bessman,J Biol Chem 241:4842-4847 (1966)). Genes from Xanthomonas campestris,Aspergillus niger and Fusarium oxysporum have been annotated as1,4-lactonase and can be utilized to catalyze the transformation of4-hydroxybutyrate to GBL (Zhang et al., Appl Microbiol Biotechnol75:1087-1094 (2007)).

Gene Accession No. GI No. Organism EU596535.1: ACC61057.1 183238971Fusarium proliferatum 1 . . . 1206 xccb100_2516 YP_001903921.1 188991911Xanthomonas campestris An16g06620 CAK46996.1 134083519 Aspergillus nigerBAA34062 BAA34062.1 3810873 Fusarium oxysporum

EXAMPLE V Pathway for Conversion of Succinyl-CoA to MAA Via3-Hydroxyisobutyrate

This example describes an exemplary MAA synthesis pathway fromsuccinyl-CoA to methacrylic acid via 3-hydroxyisobutyrate.

One exemplary pathway for MAA synthesis proceeds from succinyl-CoA (seeFIG. 3). This pathway uses at least three and at most five enzymaticsteps to form MAA from succinyl-CoA. In this pathway (see FIG. 3),succinyl-CoA is first converted to (R)-methylmalonyl-CoA, which ispotentially converted to (S)-methylmalonyl-CoA by an epimerase. Eitherthe (R)- or (S)-stereoisomer of methylmalonyl-CoA is then reduced to(R)- or (S)-3-hydroxyisobutyrate, respectively, by either a pair ofenzymes (as shown in FIG. 3) or a single enzyme that exhibits acyl-CoAreductase and alcohol dehydrogenase activities. The pathway fromsuccinyl-CoA to 3-hydroxyisobutyrate has also been described in WO2007/141208. In the final step, 3-hydroxyisobutyrate is dehydrated toform MAA.

Successfully engineering this pathway involves identifying anappropriate set of enzymes with sufficient activity and specificity.This entails identifying an appropriate set of enzymes, cloning theircorresponding genes into a production host, optimizing fermentationconditions, and assaying for product formation following fermentation.To engineer a production host for the production of methacrylic acid,one or more exogenous DNA sequence(s) are expressed in microorganisms.In addition, the microorganisms can have endogenous gene(s) functionallydeleted. These modifications allow the production of methacrylic acidusing renewable feedstock.

Below and herein are described a number of biochemically characterizedcandidate genes capable of encoding enzymes that catalyze each step ofthe desired pathway. Although described using E. coli as a host organismto engineer the pathway, essentially any suitable host organism can beused. Specifically listed are genes that are native to E. coli as wellas genes in other organisms that can be applied to catalyze theappropriate transformations when properly cloned and expressed.

Referring to FIG. 3, step 1 involves methylmalonyl-CoA mutase (EC5.4.99.2). In the first step, succinyl-CoA is converted intomethylmalonyl-CoA by methylmalonyl-CoA mutase (MCM). Methylmalonyl-CoAmutase is a cobalamin-dependent enzyme that converts succinyl-CoA tomethylmalonyl-CoA. In E. coli, the reversibleadenosylcobalamin-dependant mutase participates in a three-step pathwayleading to the conversion of succinate to propionate (Haller et al.,Biochemistry 39:4622-4629 (2000)). Overexpression of the MCM genecandidate along with the deletion of YgfG can be used to prevent thedecarboxylation of methylmalonyl-CoA to propionyl-CoA and to maximizethe methylmalonyl-CoA available for MAA synthesis. MCM is encoded bygenes scpA in Escherichia coli (Bobik and Rasche, Anal. Bioanal. Chem.375:344-349 (2003); Haller et al., Biochemistry 39:4622-4629 (2000)) andmutA in Homo sapiens (Padovani and Banerjee, Biochemistry 45:9300-9306(2006)). In several other organisms MCM contains alpha and beta subunitsand is encoded by two genes. Exemplary gene candidates encoding thetwo-subunit protein are Propionibacterium fredenreichii sp. shermanimutA and mutB (Korotkova and Lidstrom, J. Biol. Chem. 279:13652-13658(2004)), Methylobacterium extorquens mcmA and mcmB (Korotkova andLidstrom, supra, 2004), and Ralstonia eutropha sbm1 and sbm2 (Peplinskiet al., Appl. Microbiol. Biotech. 88:1145-59 (2010)). Additional enzymecandidates identified based on high homology to the E. coli spcA geneproduct are also listed below.

Protein GenBank ID GI Number Organism scpA NP_417392.1 16130818Escherichia coli K12 mutA P22033.3 67469281 Homo sapiens mutA P11652.3127549 Propionibacterium fredenreichii sp. shermanii mutB P11653.3127550 Propionibacterium fredenreichii sp. shermanii mcmA Q84FZ175486201 Methylobacterium extorquens mcmB Q6TMA2 75493131Methylobacterium extorquens Sbm1 YP_724799.1 113866310 Ralstoniaeutropha H16 Sbm2 YP_726418.1 113867929 Ralstonia eutropha H16 sbmNP_838397.1 30064226 Shigella flexneri

Protein GenBank ID GI Number Organism SARI_04585 ABX24358.1 160867735Salmonella enterica YfreA_01000861 ZP_00830776.1 77975240 Yersiniafrederiksenii

These sequences can be used to identify homologue proteins in GenBank orother databases through sequence similarity searches (for example,BLASTp). The resulting homologue proteins and their corresponding genesequences provide additional exogenous DNA sequences for transformationinto E. coli or other suitable host microorganisms to generateproduction hosts. Additional gene candidates include the following,which were identified based on high homology to the E. coli spcA geneproduct.

There further exists evidence that genes adjacent to themethylmalonyl-CoA mutase catalytic genes contribute to maximum activity.For example, it has been demonstrated that the meaB gene from M.extorquens forms a complex with methylmalonyl-CoA mutase, stimulates invitro mutase activity, and possibly protects it from irreversibleinactivation (Korotkova and Lidstrom, J. Biol. Chem. 279:13652-13658(2004)). The M. extorquens meaB gene product is highly similar to theproduct of the E. coli argK gene (BLASTp: 45% identity, e-value: 4e-67),which is adjacent to scpA on the chromosome. No sequence for a meaBhomolog in P. freudenreichii is catalogued in GenBank. However, thePropionibacterium acnes KPA171202 gene product, YP_(—)055310.1, is 51%identical to the M. extorquens meaB protein and its gene is alsoadjacent to the methylmalonyl-CoA mutase gene on the chromosome. Asimilar gene is encoded by H16_B1839 of Ralstonia eutropha H16.

Gene GenBank ID GI Number Organism argK AAC75955.1 1789285 Escherichiacoli K12 PPA0597 YP_055310.1 50842083 Propionibacterium acnes KPA1712022QM8_B 158430328 Methylobacterium extorquens H16_B1839 YP_841351.1116695775 Ralstonia eutropha H16

E. coli can synthesize adenosylcobalamin, a necessary cofactor for thisreaction, only when supplied with the intermediates cobinamide orcobalamin (Lawrence and Roth. J. Bacteriol. 177:6371-6380 (1995);Lawrence and Roth, Genetics 142:11-24 (1996)). Alternatively, theability to synthesize cobalamins de novo has been conferred upon E. colifollowing the expression of heterologous genes (Raux et al., J.Bacteriol. 178:753-767 (1996)).

Referring to FIG. 3, step 2 involves methylmalonyl-CoA epimerase (EC5.1.99.1). The second enzyme in the pathway, methylmalonyl-CoA epimerase(MMCE), interconverts (R)-methylmalonyl-CoA and (S)-methylmalonyl-CoA.MMCE is an essential enzyme in the breakdown of odd-numbered fatty acidsand of the amino acids valine, isoleucine, and methionine.Methylmalonyl-CoA epimerase activity is not believed to be encoded inthe E. coli genome (Boynton et al., J. Bacteriol. 178:3015-3024 (1996)),but is present in other organisms such as Homo sapiens (YqjC) (Fullerand Leadlay, Biochem. J. 213:643-650 (1983)), Rattus norvegicus (Mcee)(Bobik and Rasche, J. Biol. Chem. 276:37194-37198 (2001)),Propionibacterium shermanii (AF454511) (Fuller. and Leadlay, Biochem. J.213:643-650 (1983); Haller et al., Biochemistry 39:4622-4629 (2000);McCarthy et al., Structure 9:637-646.2001)) and Caenorhabditis elegans(mmce) (Kuhnl et al., FEBS J. 272:1465-1477 (2005)). An additional genecandidate, AE016877 in Bacillus cereus, has high sequence homology toother characterized enzymes. This enzymatic step may or may not benecessary depending upon the stereospecificity of the enzyme or enzymesused for the conversion of methylmalonyl-CoA to 3-hydroxyisobutyrate(steps 3-4 in FIG. 3). These genes/proteins are described below.

Gene GenBank ID GI Number Organism YqjC NP_390273 255767522 Bacillussubtilis MCEE Q96PE7.1 50401130 Homo sapiens Mcee_predictedNP_001099811.1 157821869 Rattus norvegicus AF454511 AAL57846.1 18042135Propionibacterium fredenreichii sp. shermanii mmce AAT92095.1 51011368Caenorhabditis elegans AE016877 AAP08811.1 29895524 Bacillus cereus ATCC14579

Referring to FIG. 3, step 3 involves methylmalonyl-CoA reductase (EC1.2.1.-). As shown in FIG. 3, the reduction of methylmalonyl-CoA to itscorresponding alcohol, 3-hydroxyisobutyrate, can proceed by twoenzymatic steps. The first step, conversion of methylmalonyl-CoA tomethylmalonic semialdehyde, is accomplished by a CoA-dependent aldehydedehydrogenase. An enzyme encoded by a malonyl-CoA reductase gene fromSulfolobus tokodaii (Alber et. al., J. Bacteriol. 188(24):8551-8559(2006)), has been shown to catalyze the conversion of methylmalonyl-CoAto its corresponding aldehyde (WO2007141208). A similar enzyme exists inMetallosphaera sedula (Alber et. al., J. Bacteriol. 188(24):8551-8559(2006)). Several additional CoA dehydrogenases are capable also ofreducing an acyl-CoA to its corresponding aldehyde. The reduction ofmethylmalonyl-CoA to its corresponding aldehyde, methylmalonatesemialdehyde, is catalyzed by a CoA-dependent aldehyde dehydrogenase.Exemplary enzymes include fatty acyl-CoA reductase, succinyl-CoAreductase (EC 1.2.1.76), acetyl-CoA reductase and butyryl-CoA reductase.Exemplary fatty acyl-CoA reductase enzymes are encoded by acyl ofAcinetobacter calcoaceticus (Reiser and Somerville. J. Bacteriol.179:2969-2975 (1997)), and Acinetobacter sp. M-1 fatty acyl-CoAreductase (Ishige et al., Appl. Environ. Microbiol. 68:1192-1195(2002)). Also known is a CoA- and NADP-dependent succinate semialdehydedehydrogenase (also referred to as succinyl-CoA reductase) encoded bythe sucD gene in Clostridium kluyveri (Sohling and Gottschalk, J.Bacteriol. 178:871-880 (1996); Sohling and Gottschalk, J. Bacteriol.178:871-880 (1996)) and sucD of P. gingivalis (Takahashi, J. Bacteriol182:4704-4710 (2000)). Additional succinyl-CoA reductase enzymesparticipate in the 3-hydroxypropionate/4-hydroxybutyrate cycle ofthermophilic archaea including Metallosphaera sedula (Berg et al.,Science 318:1782-1786 (2007)) and Thermoproteus neutrophilus (Ramos-Veraet al., J Bacteriol, 191:4286-4297 (2009)). The M. sedula enzyme,encoded by Msed_(—)0709, is strictly NADPH-dependent and also hasmalonyl-CoA reductase activity. The T. neutrophilus enzyme is activewith both NADPH and NADH. The enzyme acylating acetaldehydedehydrogenase in Pseudomonas sp, encoded by bphG, is also a goodcandidate as it has been demonstrated to oxidize and acylateacetaldehyde, propionaldehyde, butyraldehyde, formaldehyde and thebranched-chain compound isobutyraldehyde (Powlowski et al., J.Bacteriol. 175:377-385 (1993)). In addition to reducing acetyl-CoA toethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides hasbeen shown to oxidize the branched chain compound isobutyraldehyde toisobutyryl-CoA (Kazahaya, J. Gen. Appl. Microbiol. 18:43-55 (1972); andKoo et al., Biotechnol Lett. 27:505-510 (2005)). Butyraldehydedehydrogenase catalyzes a similar reaction, conversion of butyryl-CoA tobutyraldehyde, in solventogenic organisms such as Clostridiumsaccharoperbutylacetonicum (Kosaka et al., Biosci Biotechnol Biochem.,71:58-68 (2007)).

Protein GenBank ID GI Number Organism acr1 YP_047869.1 50086359Acinetobacter calcoaceticus acr1 AAC45217 1684886 Acinetobacter baylyiacr1 BAB85476.1 18857901 Acinetobacter sp. Strain M-1 MSED_0709YP_001190808.1 146303492 Metallosphaera sedula Tneu_0421 Thermoproteusneutrophilus sucD P38947.1 172046062 Clostridium kluyveri sucDNP_904963.1 34540484 Porphyromonas gingivalis bphG BAA03892.1 425213Pseudomonas sp adhE AAV66076.1 55818563 Leuconostoc mesenteroides bldAAP42563.1 31075383 Clostridium saccharoper- butylacetonicum

An additional enzyme type that converts an acyl-CoA to its correspondingaldehyde is malonyl-CoA reductase which transforms malonyl-CoA tomalonic semialdehyde. Malonyl-CoA reductase is a key enzyme inautotrophic carbon fixation via the 3-hydroxypropionate cycle inthermoacidophilic archaeal bacteria (Berg, Science 318:1782-1786 (2007);and Thauer, Science 318:1732-1733 (2007)). The enzyme utilizes NADPH asa cofactor and has been characterized in Metallosphaera and Sulfolobussp. (Alber et al., J. Bacteriol. 188:8551-8559 (2006); and Hugler, J.Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded by Msed_(—)0709in Metallosphaera sedula (Alber et al., J. Bacteriol. 188:8551-8559(2006); and Berg, Science 318:1782-1786 (2007)). A gene encoding amalonyl-CoA reductase from Sulfolobus tokodaii was cloned andheterologously expressed in E. coli (Alber et al., J. Bacteriol188:8551-8559 (2006). This enzyme has also been shown to catalyze theconversion of methylmalonyl-CoA to its corresponding aldehyde(WO2007141208 (2007)). Although the aldehyde dehydrogenase functionalityof these enzymes is similar to the bifunctional dehydrogenase fromChloroflexus aurantiacus, there is little sequence similarity. Bothmalonyl-CoA reductase enzyme candidates have high sequence similarity toaspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reductionand concurrent dephosphorylation of aspartyl-4-phosphate to aspartatesemialdehyde. Additional gene candidates can be found by sequencehomology to proteins in other organisms including Sulfolobussolfataricus and Sulfolobus acidocaldarius and have been listed below.Yet another candidate for CoA-acylating aldehyde dehydrogenase is theald gene from Clostridium beijerinckii (Toth, Appl. Environ. Microbiol.65:4973-4980 (1999). This enzyme has been reported to reduce acetyl-CoAand butyryl-CoA to their corresponding aldehydes. This gene is verysimilar to eutE that encodes acetaldehyde dehydrogenase of Salmonellatyphimurium and E. coli (Toth, Appl. Environ. Microbiol. 65:4973-4980(1999).

Gene GenBank ID GI Number Organism Msed_0709 YP_001190808.1 146303492Metallosphaera sedula mcr NP_378167.1 15922498 Sulfolobus tokodaii asd-2NP_343563.1 15898958 Sulfolobus solfataricus Saci_2370 YP_256941.170608071 Sulfolobus acidocaldarius Ald AAT66436 49473535 Clostridiumbeijerinckii eutE AAA80209 687645 Salmonella typhimurium eutE P774452498347 Escherichia coli

A bifunctional enzyme with acyl-CoA reductase and alcohol dehydrogenaseactivity can directly convert methylmalonyl-CoA to 3-hydroxyisobutyrate.Exemplary bifunctional oxidoreductases that convert an acyl-CoA toalcohol include those that transform substrates such as acetyl-CoA toethanol (for example, adhE from E. coli (Kessler et al., FEBS. Lett.281:59-63 (1991))) and butyryl-CoA to butanol (for example, adhE2 fromC. acetobutylicum (Fontaine et al., J. Bacteriol. 184:821-830 (2002)).The C. acetobutylicum enzymes encoded by bdh I and bdh II (Walter, etal., J. Bacteriol. 174:7149-7158 (1992)), reduce acetyl-CoA andbutyryl-CoA to ethanol and butanol, respectively. In addition toreducing acetyl-CoA to ethanol, the enzyme encoded by adhE inLeuconostoc mesenteroides has been shown to oxide the branched chaincompound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al., J. Gen.Appl. Microbiol. 18:43-55 (1972); Koo et al., Biotechnol Lett,27:505-510 (2005)). Another exemplary enzyme can convert malonyl-CoA to3-HP. An NADPH-dependent enzyme with this activity has characterized inChloroflexus aurantiacus where it participates in the3-hydroxypropionate cycle (Hugler et al., J Bacteriol, 184:2404-2410(2002); Strauss et al., Eur J Biochem, 215:633-643 (1993)). This enzyme,with a mass of 300 kDa, is highly substrate-specific and shows littlesequence similarity to other known oxidoreductases (Hugler et al.,supra). No enzymes in other organisms have been shown to catalyze thisspecific reaction; however there is bioinformatic evidence that otherorganisms may have similar pathways (Klatt et al., Env Microbiol,9:2067-2078 (2007)). Enzyme candidates in other organisms includingRoseiflexus castenholzii, Erythrobacter sp. NAP1 and marine gammaproteobacterium HTCC2080 can be inferred by sequence similarity.

Protein GenBank ID GI Number Organism adhE NP_415757.1 16129202Escherichia coli adhE2 AAK09379.1 12958626 Clostridium acetobutylicumadhE AAV66076.1 55818563 Leuconostoc mesenteroides bdh I NP_349892.115896543 Clostridium acetobutylicum bdh II NP_349891.1 15896542Clostridium acetobutylicum mcr AAS20429.1 42561982 Chloroflexusaurantiacus Rcas_2929 YP_001433009.1 156742880 Roseiflexus castenholziiNAP1_02720 ZP_01039179.1 85708113 Erythrobacter sp. NAP1 MGP2080_00535ZP_01626393.1 119504313 marine gamma proteobacterium HTCC2080

Referring to FIG. 3, step 4 involves 3-hydroxyisobutyrate dehydrogenase(EC 1.1.1.31). 3-hydroxyisobutyrate dehydrogenase catalyzes thereversible oxidation of 3-hydroxyisobutyrate to methylmalonatesemialdehyde. The reduction of methylmalonate semialdehyde to3-hydroxyisobutyrate is catalyzed by methylmalonate semialdehydereductase or 3-hydroxyisobutyrate dehydrogenase. This enzymeparticipates in valine, leucine and isoleucine degradation and has beenidentified in bacteria, eukaryotes, and mammals. The enzyme encoded byP84067 from Thermus thermophilus HB8 has been structurally characterized(Lokanath et al., J. Mol. Biol. 352:905-917 (2005)). The reversibilityof the human 3-hydroxyisobutyrate dehydrogenase was demonstrated usingisotopically-labeled substrate (Manning and Pollitt, Biochem. J.231:481-484 (1985)). Additional genes encoding this enzyme include 3hidhin Homo sapiens (Hawes et al., Methods Enzymol. 324:218-228 (2000)) andOryctolagus cuniculus (Chowdhury et al., Biosci. Biotechnol. Biochem.60:2043-2047 (1996); Hawes et al., Methods Enzymol. 324:218-228 (2000)),mmsb in Pseudomonas aeruginosa, and dhat in Pseudomonas putida (Aberhartand Hsu. J. Chem. Soc. [Perkin 1] 6:1404-1406 (1979); Chowdhury et al.,Biosci. Biotechnol. Biochem. 67:438-441 (2003); Chowdhury et al.,Biosci. Biotechnol. Biochem. 60:2043-2047 (1996)). Several3-hydroxyisobutyrate dehydrogenase enzymes have been characterized inthe reductive direction, including mmsB from Pseudomonas aeruginosa(Gokarn et al., U.S. Pat. No. 7,393,676 (2008)) and mmsB fromPseudomonas putida.

PROTEIN GENBANK ID GI NUMBER ORGANISM P84067 P84067 75345323 Thermusthermophilus 3hidh P31937.2 12643395 Homo sapiens 3hidh P32185.1 416872Oryctolagus cuniculus mmsB NP_746775.1 26991350 Pseudomonas putida mmsBP28811.1 127211 Pseudomonas aeruginosa dhat Q59477.1 2842618 Pseudomonasputida

Referring to FIG. 3, as an alternative, steps 3 and 4 can involve acombined Alcohol/Aldehyde dehydrogenase (EC 1.2.1.-). Methylmalonyl-CoAcan be reduced to 3-hydroxyisobutyrate in one step by a multifunctionalenzyme with dual acyl-CoA reductase and alcohol dehydrogenase activity.Although the direct conversion of methylmalonyl-CoA to3-hydroxyisobutyrate has not been reported, this reaction is similar tothe common conversions such as acetyl-CoA to ethanol and butyryl-CoA tobutanol, which are catalyzed by CoA-dependant enzymes with both alcoholand aldehyde dehydrogenase activities. Gene candidates include the E.coli adhE (Kessler et al., FEBS Lett. 281:59-63 (1991)) and C.acetobutylicum bdh I and bdh II (Walter, et al., J. Bacteriol.174:7149-7158 (1992)), which can reduce acetyl-CoA and butyryl-CoA toethanol and butanol, respectively. In addition to reducing acetyl-CoA toethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides hasbeen shown to oxide the branched chain compound isobutyraldehyde toisobutyryl-CoA (Kazahaya et al., J. Gen. Appl. Microbiol. 18:43-55(1972); Koo et al., Biotechnol. Lett. 27:505-510 (2005)). An additionalcandidate enzyme for converting methylmalonyl-CoA directly to3-hydroxyisobutyrate is encoded by a malonyl-CoA reductase fromChloroflexus aurantiacus (Hagler, et al., J. Bacteriol. 184(9):2404-2410(2002).

Protein GenBank ID GI Number Organism mcr YP_001636209.1 163848165Chloroflexus aurantiacus adhE NP_415757.1 16129202 Escherichia coli bdhI NP_349892.1 15896543 Clostridium acetobutylicum bdh II NP_349891.115896542 Clostridium acetobutylicum adhE AAV66076.1 55818563 Leuconostocmesenteroides

Referring to FIG. 3, step 5 involves 3-hydroxyisobutyrate dehydratase(EC 4.2.1.-). The final step involves the dehydration of3-hydroxyisobutyrate to methacrylic acid. The dehydration of3-hydroxyisobutyrate to methylacrylic acid is catalyzed by an enzymewith 3-hydroxyisobutyrate dehydratase activity. Although no directevidence for this specific enzymatic transformation has been identified,most dehydratases catalyze the α,β-elimination of water, which involvesactivation of the α-hydrogen by an electron-withdrawing carbonyl,carboxylate, or CoA-thiol ester group and removal of the hydroxyl groupfrom the β-position (Buckel and Barker, J Bacteriol. 117:1248-1260(1974); Martins et al, Proc. Natl. Acad. Sci. USA 101:15645-15649(2004)). This is the exact type of transformation proposed for the finalstep in the methacrylate pathway. In addition, the proposedtransformation is highly similar to the 2-(hydroxymethyl)glutaratedehydratase of Eubacterium barkeri, which can catalyze the conversion of2-hydroxymethyl glutarate to 2-methylene glutarate. This enzyme has beenstudied in the context of nicotinate catabolism and is encoded by hmd(Alhapel et al., Proc. Natl. Acad. Sci. USA 103:12341-12346 (2006)).Similar enzymes with high sequence homology are found in Bacteroidescapillosus, Anaerotruncus colihominis, and Natranaerobius thermophilius.Several enzymes are known to catalyze the alpha, beta elimination ofhydroxyl groups. Exemplary enzymes include 2-(hydroxymethyl)glutaratedehydratase (EC 4.2.1.-), fumarase (EC 4.2.1.2), 2-keto-4-pentenoatedehydratase (EC 4.2.1.80), citramalate hydrolyase and dimethylmaleatehydratase.

2-(Hydroxymethyl)glutarate dehydratase is a [4Fe-4S]-containing enzymethat dehydrates 2-(hydroxymethyl)glutarate to 2-methylene-glutarate,studied for its role in nicontinate catabolism in Eubacterium barkeri(formerly Clostridium barkeri) (Alhapel et al., Proc Natl Acad Sci USA103:12341-12346 (2006)). Similar enzymes with high sequence homology arefound in Bacteroides capillosus, Anaerotruncus colihominis, andNatranaerobius thermophilius. These enzymes are also homologous to theα- and β-subunits of [4Fe-4S]-containing bacterial serine dehydratases,for example, E. coli enzymes encoded by tdcG, sdhB, and sdaA). An enzymewith similar functionality in E. barkeri is dimethylmaleate hydratase, areversible Fe2+-dependent and oxygen-sensitive enzyme in the aconitasefamily that hydrates dimethylmaeate to form (2R,3S)-2,3-dimethylmalate.This enzyme is encoded by dmdAB (Alhapel et al., Proc Natl Acad Sci USA103:12341-6 (2006); Kollmann-Koch et al., Hoppe Seylers. Z. PhysiolChem. 365:847-857 (1984)).

Protein GenBank ID GI Number Organism hmd ABC88407.1 86278275Eubacterium barkeri BACCAP_02294 ZP_02036683.1 154498305 Bacteroidescapillosus ANACOL_02527 ZP_02443222.1 167771169 Anaerotruncuscolihominis NtherDRAFT_2368 ZP_02852366.1 169192667 Natranaerobiusthermophilus dmdA ABC88408 86278276 Eubacterium barkeri dmdB ABC8840986278277 Eubacterium barkeri

Fumarate hydratase enzymes, which naturally catalyze the reversiblehydration of fumarate to malate. Although the ability of fumaratehydratase to react on branched substrates with 3-oxobutanol as asubstrate has not been described, a wealth of structural information isavailable for this enzyme and other researchers have successfullyengineered the enzyme to alter activity, inhibition and localization(Weaver, Acta Crystallogr. D Biol. Crystallogr. 61:1395-1401 (2005)). E.coli has three fumarases: FumA, FumB, and FumC that are regulated bygrowth conditions. FumB is oxygen sensitive and only active underanaerobic conditions. FumA is active under microanaerobic conditions,and FumC is the only active enzyme in aerobic growth (Tseng et al., J.Bacteriol. 183:461-467 (2001); Woods et al., Biochm. Biophys. Acta954:14-26 (1988); Guest et al., J Gen Microbiol 131:2971-2984 (1985)).Exemplary enzyme candidates include those encoded by fumC fromEscherichia coli (Estevez et al., Protein Sci. 11:1552-1557 (2002); Hongand Lee, Biotechnol. Bioprocess Eng. 9:252-255 (2004); Rose and Weaver,Proc. Natl. Acad. Sci. USA 101:3393-3397 (2004)), and enzymes found inCampylobacter jejuni (Smith et al., Int. J. Biochem. Cell Biol.31:961-975 (1999)), Thermus thermophilus (Mizobata et al., Arch.Biochem. Biophys. 355:49-55 (1998)), and Rattus norvegicus (Kobayashi etal., J. Biochem. 89:1923-1931 (1981)). Similar enzymes with highsequence homology include fum1 from Arabidopsis thaliana and fumC fromCorynebacterium glutamicum. The MmcBC fumarase from Pelotomaculumthermopropionicum is another class of fumarase with two subunits(Shimoyama et al., FEMS Microbiol Lett. 270:207-213 (2007)).

Protein GenBank ID GI Number Organism fumA NP_416129.1 16129570Escherichia coli fumB NP_418546.1 16131948 Escherichia coli fumCNP_416128.1 16129569 Escherichia coli fumC O69294 9789756 Campylobacterjejuni

Protein GenBank ID GI Number Organism fumC P84127 75427690 Thermusthermophilus fumH P14408 120605 Rattus norvegicus fum1 P93033 39931311Arabidopsis thaliana fumC Q8NRN8 39931596 Corynebacterium glutamicumMmcB YP_001211906 147677691 Pelotomaculum thermopropionicum MmcCYP_001211907 147677692 Pelotomaculum thermopropionicum

Dehydration of 4-hydroxy-2-oxovalerate to 2-oxopentenoate is catalyzedby 4-hydroxy-2-oxovalerate hydratase (EC 4.2.1.80). This enzymeparticipates in aromatic degradation pathways and is typicallyco-transcribed with a gene encoding an enzyme with4-hydroxy-2-oxovalerate aldolase activity. Exemplary gene products areencoded by mhpD of E. coli (Ferrandez et al., J Bacteriol. 179:2573-2581(1997); Pollard et al., Eur J Biochem. 251:98-106 (1998)), todG and cmtFof Pseudomonas putida (Lau et al., Gene 146:7-13 (1994); Eaton, J.Bacteriol. 178:1351-1362 (1996)), cnbE of Comamonas sp. CNB-1 (Ma etal., Appl Environ Microbiol 73:4477-4483 (2007)) and mhpD ofBurkholderia xenovorans (Wang et al., FEBS J 272:966-974 (2005)). Aclosely related enzyme, 2-oxohepta-4-ene-1,7-dioate hydratase,participates in 4-hydroxyphenylacetic acid degradation, where itconverts 2-oxo-hept-4-ene-1,7-dioate (OHED) to2-oxo-4-hydroxy-hepta-1,7-dioate using magnesium as a cofactor (Burks etal., J. Am. Chem. Soc. 120: (1998)). OHED hydratase enzyme candidateshave been identified and characterized in E. coli C (Roper et al., Gene156:47-51 (1995); Izumi et al., J Mol. Biol. 370:899-911 (2007)) and E.coli W (Prieto et al., J Bacteriol. 178:111-120 (1996)). Sequencecomparison reveals homologs in a wide range of bacteria, plants andanimals. Enzymes with highly similar sequences are contained inKlebsiella pneumonia (91% identity, eval=2e-138) and Salmonella enterica(91% identity, eval=4e-138), among others.

GenBank Gene Accession No. GI No. Organism mhpD AAC73453.2 87081722Escherichia coli cmtF AAB62293.1 1263188 Pseudomonas putida todGAAA61942.1 485738 Pseudomonas putida cnbE YP_001967714.1 190572008Comamonas sp. CNB-1 mhpD Q13VU0 123358582 Burkholderia xenovorans hpcGCAA57202.1 556840 Escherichia coli C hpaH CAA86044.1 757830 Escherichiacoli W hpaH ABR80130.1 150958100 Klebsiella pneumoniae Sari_01896ABX21779.1 160865156 Salmonella enterica

Another enzyme candidate is citramalate hydrolyase (EC 4.2.1.34), anenzyme that naturally dehydrates 2-methylmalate to mesaconate. Thisenzyme has been studied in Methanocaldococcus jannaschii in the contextof the pyruvate pathway to 2-oxobutanoate, where it has been shown tohave a broad substrate specificity (Drevland et al., J Bacteriol.189:4391-4400 (2007)). This enzyme activity was also detected inClostridium tetanomorphum, Morganella morganii, Citrobacter amalonaticuswhere it is thought to participate in glutamate degradation (Kato etal., Arch. Microbiol 168:457-463 (1997)). The M. jannaschii proteinsequence does not bear significant homology to genes in these organisms.

Protein GenBank ID GI Number Organism leuD Q58673.1 3122345Methanocaldococcus jannaschii

Dimethylmaleate hydratase (EC 4.2.1.85) is a reversible Fe²⁺-dependentand oxygen-sensitive enzyme in the aconitase family that hydratesdimethylmaeate to form (2R,3S)-2,3-dimethylmalate. This enzyme isencoded by dmdAB in Eubacterium barkeri (Alhapel et al., supra;Kollmann-Koch et al., Hoppe Seylers. Z. Physiol Chem. 365:847-857(1984)).

Protein GenBank ID GI Number Organism dmdA ABC88408 86278276 Eubacteriumbarkeri dmdB ABC88409.1 86278277 Eubacterium barkeri

This example describes a biosynthetic pathway for production of MMA fromsuccinyl-CoA.

EXAMPLE VI Pathway for Conversion of Succinyl-CoA to MAA via3-Amino-2-Methylpropanoate

This example describes an exemplary MAA synthesis pathway fromsuccinyl-CoA to MAA via 3-amino-methylpropanoate.

Another exemplary pathway for MAA biosynthesis proceeds fromsuccinyl-CoA through 3-amino-2-methylpropanoate (see FIG. 4). The firstthree steps of this pathway, involving the conversion of succinyl-CoA tomethylmalonate semialdehyde, are identical to the succinyl-CoA to MAApathway described in Example V (see FIG. 3). The pathway diverges atstep 4, where methylmalonate semialdehyde is converted to3-amino-2-methylpropionate by a transaminase. The final pathway stepentails deamination of 3-amino-2-methylpropionate to methacrylic acid.

Enzyme and gene candidates for catalyzing the first three pathway stepsare described in Example V. Gene candidates for steps 4 and 5 arediscussed below.

Referring to FIG. 4, step 4 involves 3-amino-2-methylpropionatetransaminase (EC 2.6.1.22). 3-amino-2-methylpropionate transaminasecatalyzes the transformation from methylmalonate semialdehyde to3-amino-2-methylpropionate. The enzyme, characterized in Rattusnorvegicus and Sus scrofa and encoded by Abat, has been shown tocatalyze this transformation in the direction of interest in the pathway(Kakimoto et al., Biochim. Biophys. Acta 156:374-380 (1968); Tamaki etal., Methods Enzymol. 324:376-389 (2000)). Enzyme candidates in otherorganisms with high sequence homology to 3-amino-2-methylpropionatetransaminase include Gta-1 in C. elegans and gabT in Bacillus subtilus.Additionally, one of the native GABA aminotransferases in E. coli,encoded by gene gabT, has been shown to have broad substrate specificityand may utilize 3-amino-2-methylpropionate as a substrate (Liu et al.,Biochemistry 43:10896-10905 (2004); Schulz et al., Appl. Environ.Microbiol. 56:1-6 (1990)).

Protein GenBank ID GI Number Organism Abat P50554.3 122065191 Rattusnorvegicus Abat P80147.2 120968 Sus scrofa Gta-1 Q21217.1 6016091Caenorhabditis elegans gabT P94427.1 6016090 Bacillus subtilus gabTP22256.1 16130576 Escherichia coli K12

Referring to FIG. 4, step 5 involves 3-amino-2-methylpropionate ammonialyase (EC 4.3.1.-). In the final step of this pathway,3-amino-2-methylpropionate is deaminated to methacrylic acid. An enzymecatalyzing this exact transformation has not been demonstratedexperimentally; however the native E. coli enzyme, aspartate ammonialyase (EC 4.3.1.1), may be able to catalyze this reaction. Encoded byaspA in E. coli, aspartate ammonia lyase deaminates asparatate to formfumarate but can also react with alternate substratesaspartatephenylmethylester, asparagine, benzyl-aspartate and malate (Maet al., Ann. N.Y. Acad. Sci. 672:60-65 (1992)). In a separate study,directed evolution was been employed on this enzyme to alter substratespecificity (Asano et al., Biomol. Eng. 22:95-101 (2005)). Genesencoding aspartase in other organisms include ansB in Bacillus subtilus(Sjostrom et al., Biochim. Biophys. Acta 1324:182-190 (1997)) and aspAin Pseudomonas fluorescens (Takagi et al., J. Biochem. 96:545-552(1984); Takagi et al., J. Biochem. 100:697-705 (1986)) and Serratiamarcescens (Takagi et al., J. Bacteriol. 161:1-6 (1985)).

Protein GenBank ID GI Number Organism aspA P0AC38.1 90111690 Escherichiacoli K12 ansB P26899.1 251757243 Bacillus subtilus aspA P07346.1 114273Pseudomonas fluorescens aspA P33109.1 416661 Serratia marcescens

This example describes an MAA biosynthetic pathway from succinyl-CoA.

EXAMPLE VII Pathway for Conversion of 4-Hydroxybutyryl-CoA to3-Hydroxyisobutyric Acid or MAA

This example describes an exemplary 3-hydroxyisobutyric acid or MAAsynthesis pathway from 4-hydroxybutyryl-CoA.

An additional exemplary pathway entails the conversion of 4HB-CoA to MAA(see FIG. 5). In the first step, 4HB-CoA is converted to3-hydroxyisobutyryl-CoA (3-Hib-CoA) by a methylmutase. 3-Hib-CoA canthen be converted to 3-hydroxyisobutyrate by a CoA hydrolase, synthaseor transferase. 3-hydroxyisobutyrate can be secreted and recovered as aproduct or as a final step in the production of methacrylic acid.3-Hydroxybutyrate can be dehydrated to form methacrylic acid.Alternatively, 3-Hib-CoA can be dehydrated to methacrylyl-CoA which isthen converted to MAA by a hydrolase, synthase, or transferase. Theenzymes required for converting the tricarboxylic acid cycleintermediates, alpha-ketoglutarate, succinate, or succinyl-CoA, into4HB-CoA, are well-documented (Burk et al., U.S. application Ser. No.12/049,256, filed Mar. 14, 2008; Lutke-Eversloh and Steinbuchel. FEMSMicrobiol. Lett. 181:63-71 (1999); Sohling and Gottschalk, Eur. J.Biochem. 212:121-127 (1993); Sohling and Gottschalk, J. Bacteriol.178:871-880 (1996); Valentin et al., Eur. J. Biochem. 227:43-60 (1995);Wolff and Kenealy, Protein Expr. Pur 6:206-212. (1995)).

Referring to FIG. 5, step 1 involves 4-hydroxybutyryl-CoA mutase (EC5.4.99.-). The conversion of 4HB-CoA to 3-hydroxyisobutyryl-CoA has yetto be demonstrated experimentally. However, two methylmutases, that is,isobutyryl-CoA mutase (ICM) and methylmalonyl-CoA mutase (MCM), whichcatalyze similar reactions, are good candidates given the structuralsimilarity of their corresponding substrates. Methylmalonyl-CoA mutaseis a cobalamin-dependent enzyme that converts succinyl-CoA tomethylmalonyl-CoA. This enzyme and suitable gene candidates werediscussed in the succinyl-CoA to MAA pathway (see Example V).

Alternatively, isobutyryl-CoA (ICM, EC 5.4.99.13) could catalyze theproposed transformation. ICM is a cobalamin-dependent methylmutase inthe MCM family that reversibly rearranges the carbon backbone ofbutyryl-CoA into isobutyryl-CoA (Ratnatilleke et al., J. Biol. Chem.274:31679-31685 (1999)). A recent study of a novel ICM in Methylibiumpetroleiphilum, along with previous work, provides evidence thatchanging a single amino acid near the active site alters the substratespecificity of the enzyme (Ratnatilleke et al., J. Biol. Chem.274:31679-31685 (1999); Rohwerder et al., Appl. Environ. Microbiol.72:4128-4135. (2006)). This indicates that, if a native enzyme is unableto catalyze or exhibits low activity for the conversion of 4HB-CoA to3HIB-CoA, the enzyme can be rationally engineered to increase thisactivity. Exemplary ICM genes encoding homodimeric enzymes include icmAin Streptomyces coelicolor A3 (Alhapel et al., Proc. Natl. Acad. Sci.USA 103:12341-12346 (2006)) and Mpe_B0541 in Methylibium petroleiphilumPM1 (Ratnatilleke et al., J. Biol. Chem. 274:31679-31685 (1999);Rohwerder et al., Appl. Environ. Microbiol. 72:4128-4135 (2006)). Genesencoding heterodimeric enzymes include icm and icmB in Streptomycescinnamonensis (Ratnatilleke et al., J. Biol. Chem. 274:31679-31685(1999); Vrijbloed et al., J. Bacteriol. 181:5600-5605. (1999);Zerbe-Burkhardt et al., J. Biol. Chem. 273:6508-6517 (1998)). Enzymesencoded by icmA and icmB genes in Streptomyces avermitilis MA-4680 showhigh sequence similarity to known ICMs. These genes/proteins areidentified below.

Gene GenBank ID GI Number Organism icmA CAB40912.1 4585853 Streptomycescoelicolor A3(2) Mpe_B0541 YP_001023546.1 124263076 Methylibiumpetroleiphilum PM1 icm AAC08713.1 3002492 Streptomyces cinnamonensisicmB CAB59633.1 6137077 Streptomyces cinnamonensis icmA NP_824008.129829374 Streptomyces avermitilis icmB NP_824637.1 29830003 Streptomycesavermitilis

Referring to FIG. 5, step 2 involves 3-hydroxyisobutyryl-CoA hydrolase(EC 3.1.2.4), synthetase (EC 6.2.1.-) or 3-hydroxyisobutyryl-CoAtransferase (EC 2.8.3.-). Step 5 involves methacrylyl-CoA hydrolase,synthetase, or transferase. These transformations can be performed bydifferent classes of enzymes including CoA hydrolases (EC 3.1.2.-), CoAtransferases (EC 2.8.3.-), and CoA synthetases (EC 6.1.2.-). Asdiscussed earlier, pathway energetics are most favorable if a CoAtransferase or a CoA synthetase is employed to accomplish thistransformation (Table 1).

In the CoA-transferase family, E. coli enzyme acyl-CoA:acetate-CoAtransferase, also known as acetate-CoA transferase (EC 2.8.3.8), hasbeen shown to transfer the CoA moiety to acetate from a variety ofbranched and linear acyl-CoA substrates, including isobutyrate (Matthiesand Schink, Appl. Environ. Microbiol. 58:1435-1439 (1992)), valerate(Vanderwinkel et al., Biochem. Biophys. Res. Commun. 33:902-908 (1968))and butanoate (Vanderwinkel et al. supra, 1968). This enzyme is encodedby atoA (alpha subunit) and atoD (beta subunit) in E. coli sp. K12(Korolev et al., Acta Crystallogr. D Biol. Crystallogr. 58:2116-2121(2002); Vanderwinkel et al., supra, 1968) and actA and cg0592 inCorynebacterium glutamicum ATCC 13032 (Duncan et al., Appl. Environ.Microbiol. 68:5186-5190 (2002)) and represents an ideal candidate tocatalyze the desired 3-hydroxyisobutyryl-CoA transferase ormethacrylyl-CoA transferase biotransformations shown in FIG. 5, steps 2and 5. Candidate genes by sequence homology include atoD and atoA inEscherichia coli UT189. Similar enzymes also exist in Clostridiumacetobutylicum and Clostridium saccharoperbutylacetonicum.

Gene GenBank ID GI Number Organism atoA P76459.1 2492994 Escherichiacoli K12 atoD P76458.1 2492990 Escherichia coli K12

Gene GenBank ID GI Number Organism actA YP_226809.1 62391407Corynebacterium glutamicum ATCC 13032 cg0592 YP_224801.1 62389399Corynebacterium glutamicum ATCC 13032 atoA ABE07971.1 91073090Escherichia coli UT189 atoD ABE07970.1 91073089 Escherichia coli UT189ctfA NP_149326.1 15004866 Clostridium acetobutylicum ctfB NP_149327.115004867 Clostridium acetobutylicum ctfA AAP42564.1 31075384 Clostridiumsaccharoperbutylacetonicum ctfB AAP42565.1 31075385 Clostridiumsaccharoperbutylacetonicum

Additional exemplary transferase transformations are catalyzed by thegene products of cat1, cat2, and cat3 of Clostridium kluyveri which havebeen shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, andbutyryl-CoA acetyltransferase activity, respectively (Sohling andGottschalk, J. Bacteriol. 178(3): 871-880 (1996); Seedorf et al., Proc.Natl. Acad. Sci. USA, 105(6):2128-2133 (2008)).

Gene GenBank ID GI Number Organism cat1 P38946.1 729048 Clostridiumkluyveri cat2 P38942.2 172046066 Clostridium kluyveri cat3 EDK35586.1146349050 Clostridium kluyveri

The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from anaerobicbacterium Acidaminococcus fermentans reacts with diacid glutaconyl-CoAand 3-butenoyl-CoA (Mack and Buckel, FEBS Lett. 405:209-212 (1997)). Thegenes encoding this enzyme are gctA and gctB. This enzyme has reducedbut detectable activity with other CoA derivatives includingglutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckelet al., Eur. J. Biochem. 118:315-321 (1981)). The enzyme has been clonedand expressed in E. coli (Mack et al., Eur. J. Biochem. 226:41-51(1994)).

Gene GenBank ID GI Number Organism gctA CAA57199.1 559392Acidaminococcus fermentans gctB CAA57200.1 559393 Acidaminococcusfermentans

Additional enzyme candidates include succinyl-CoA:3-ketoacid CoAtransferases which utilize succinate as the CoA acceptor. Exemplarysuccinyl-CoA:3:ketoacid-CoA transferases are present in Helicobacterpylori (Corthesy-Theulaz et al., J. Biol. Chem. 272:25659-25667 (1997))and Bacillus subtilis (Stols et al., Protein Expr. Purif. 53:396-403(2007)).

Gene GenBank ID GI Number Organism HPAG1_0676 YP_627417 108563101Helicobacter pylori HPAG1_0677 YP_627418 108563102 Helicobacter pyloriScoA NP_391778 16080950 Bacillus subtilis ScoB NP_391777 16080949Bacillus subtilis

A candidate ATP synthase is ADP-forming acetyl-CoA synthetase (ACD, EC6.2.1.13), an enzyme that couples the conversion of acyl-CoA esters totheir corresponding acids with the concurrent synthesis of ATP. Althoughthis enzyme has not been shown to react with 3-hydroxyisobutyryl-CoA ormethacrylyl-CoA as a substrate, several enzymes with broad substratespecificities have been described in the literature. ACD I fromArchaeoglobus fulgidus, encoded by AF1211, was shown to operate on avariety of linear and branched-chain substrates including isobutyrate,isopentanoate, and fumarate (Musfeldt and Schonheit, J. Bacteriol.184:636-644 (2002)). The enzyme from Haloarcula marismortui (annotatedas a succinyl-CoA synthetase) accepts priopionate, butyrate, andbranched-chain acids (isovalerate and isobutyrate) as substrates, andwas shown to operate in the forward and reverse directions (Brasen andSchonheit, Arch. Microbiol. 182:277-287 (2004)). The ACD encoded byPAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilumshowed the broadest substrate range of all characterized ACDs, reactingwith acetyl-CoA, isobutyryl-CoA (preferred substrate) andphenylacetyl-CoA (Brasen and Schonheit, supra, 2004). However, directedevolution or engineering can be used to modify this enzyme to operate atthe physiological temperature of the host organism. The enzymes from A.fulgidus, H. marismortui and P. aerophilum have all been cloned,functionally expressed, and characterized in E. coli (Brasen andSchonheit, supra, 2004; Musfeldt and Schonheit, J. Bacteriol.184:636-644 (2002)).

Gene GenBank ID GI Number Organism AF1211 NP_070039.1 11498810Archaeoglobus fulgidus DSM 4304 scs YP_135572.1 55377722 Haloarculamarismortui ATCC 43049 PAE3250 NP_560604.1 18313937 Pyrobaculumaerophilum str. IM2

In the CoA hydrolase family, the enzyme 3-hydroxyisobutyryl-CoAhydrolase is specific for 3-HIBCoA and has been described to efficientlycatalyze the desired transformation during valine degradation (Shimomuraet al., J. Biol. Chem. 269:14248-14253 (1994)). Genes encoding thisenzyme include hibch of Rattus norvegicus (Shimomura et al., J. Biol.Chem. 269:14248-14253 (1994); Shimomura et al., Methods Enzymol.324:229-240 (2000)) and Homo sapiens (Shimomura et al., supra, 2000).Candidate genes by sequence homology include hibch of Saccharomycescerevisiae and BC_(—)2292 of Bacillus cereus.

Gene GenBank ID GI Number Organism hibch Q5XIE6.2 146324906 Rattusnorvegicus hibch Q6NVY1.2 146324905 Homo sapiens hibch P28817.2 2506374Saccharomyces cerevisiae BC_2292 Q81DR3 29895975 Bacillus cereus

Referring to FIG. 5, step 3 involves 3-hydroxyisobutyrate dehydratase(EC 4.2.1.-). The entails dehydration of 3-hydroxyisobutyrate to MAA by3-hydroxyisobutyrate dehydratase. Gene candidates for this enzyme aredescribed in the succinyl-CoA to MAA pathway (see Example V). Alsoreferring to FIG. 5, step 4 involves 3-hydroxyisobutyryl-CoA dehydratase(EC 4.2.1.-). Dehydration of 3-hydroxyisobutyryl-CoA to methacrylyl-CoAcan be accomplished by a reversible 3-hydroxyacyl-CoA dehydratase suchas crotonase (also called 3-hydroxybutyryl-CoA dehydratase, EC 4.2.1.55)or enoyl-CoA hydratase (also called 3-hydroxyacyl-CoA dehydratase, EC4.2.1.17). These enzymes are generally reversible (Moskowitz andMerrick, Biochemistry 8:2748-2755 (1969); Durre et al., FEMS Microbiol.Rev. 17:251-262 (1995)). Exemplary genes encoding crotonase enzymes canbe found in C. acetobutylicum (Boynton, et al., J. Bacteriol.178(11):3015-3024 (1996)), C. kluyveri (Hillmer and Gottschalk, FEBSLett. 21(3):351-354 (1972)), and Metallosphaera sedula (Berg et al.,Science 318(5857) 1782-1786 (2007)) though the sequence of the lattergene is not known. Enoyl-CoA hydratases, which are involved in fattyacid beta-oxidation and/or the metabolism of various amino acids, canalso catalyze the hydration of crotonyl-CoA to form 3-hydroxybutyryl-CoA(Agnihotri and Liu, Bioorg. Med. Chem. 11(1):9-20 (2003); Roberts etal., Arch. Microbiol. 117(1):99-108 (1978); Conrad et al., J. Bacteriol.118(1):103-111 (1974)). The enoyl-CoA hydratases, phaA and phaB, of P.putida are believed to carry out the hydroxylation of double bondsduring phenylacetate catabolism (Olivera et al., Proc. Natl. Acad. Sci.USA 95:6419-6424 (1998)). The paaA and paaB from P. fluorescens catalyzeanalogous transformations (Olivera et al., supra, 1998). Lastly, anumber of Escherichia coli genes have been shown to demonstrateenoyl-CoA hydratase functionality including maoC (Park and Lee, J.Bacteriol. 185:5391-5397 (2003)), paaF (Ismail et al., Eur. J. Biochem.270:3047-3054 (2003); Park and Lee, Appl. Biochem. Biotechnol.113-116:335-346 (2004); Park and Yup, Biotechnol. Bioeng. 86:681-686.(2004)), and paaG (Ismail et al., Eur. J. Biochem. 270:3047-3054 (2003);Park and Lee, Appl. Biochem. Biotechnol. 113-116:335-346 (2004); Parkand Yup, Biotechnol. Bioeng. 86:681-686 (2004)).

Gene GenBank ID GI Number Organism crt NP_349318.1 15895969 Clostridiumacetobutylicum crt1 YP_001393856 153953091 Clostridium kluyveri DSM 555paaA NP_745427.1 26990002 Pseudomonas fluorescens paaB NP_745426.126990001 Pseudomonas fluorescens phaA ABF82233.1 106636093 Pseudomonasputida phaB ABF82234.1 106636094 Pseudomonas putida maoC NP_415905.116129348 Escherichia coli paaF NP_415911.1 16129354 Escherichia colipaaG NP_415912.1 16129355 Escherichia coli

This example describes a biosynthetic pathway for production of3-hydroxyisobutyric acid or methacrylic acid from 4-hydroxybutyryl-CoA.

EXAMPLE VIII Pathway for Conversion of Alpha-Ketoglutarate to MAA ViaThreo-3-Methylaspartate

This example describes an exemplary MAA synthesis pathway fromalpha-ketoglutarate to threo-3-methylaspartate.

Another exemplary pathway for MAA biosynthesis proceeds throughalpha-ketoglutarate, a metabolite in E. coli produced in the TCA cycle(see FIG. 6).

The first step of the pathway, catalyzed by the enzyme aspartateaminotransferase, transfers an amino group from aspartate toalpha-ketoglutarate, forming glutamate and oxaloacetate. The subsequenttwo steps include rearrangement of the carbon backbone and subsequentdeamination to form mesaconate. Enzymes catalyzing these conversions arefound in the energy-yielding fermentation of glutamate in soilClostridia and other organisms capable of fermenting amino acids (Buckeland Barker, J. Bacteriol. 117:1248-1260 (1974)). The directionality ofthe pathway in these organisms is in agreement with the directionrequired for MAA synthesis in the biopathway. The final pathway stepentails decarboxylation of mesaconate to yield methacrylic acid.

Referring to FIG. 6, step 1 involves aspartate aminotransferase (EC2.6.1.1). The first step of the pathway transfers an amino group fromaspartate to alpha-ketoglutarate, forming glutamate and oxaloacetate.The genes aspC from Escherichia coli (Yagi et al., FEBS Lett. 100:81-84(1979); Yagi et al., Methods Enzymol. 113:83-89 (1985)), AAT2 fromSaccharomyces cerevisiae (Yagi et al., J. Biochem. 92:35-43 (1982)) andASPS from Arabidopsis thaliana (de la Torre et al., Plant J. 46:414-425(2006); Kwok and Hanson, J. Exp. Bot. 55:595-604 (2004); Wilkie andWarren, Protein Expr. Purif. 12:381-389 (1998)), encode the enzyme thatcatalyzes this conversion, aspartate aminotransferase.

Gene GenBank ID GI Number Organism aspC NP_415448.1 16128895 Escherichiacoli AAT2 P23542.3 1703040 Saccharomyces cerevisiae ASP5 P46248.220532373 Arabidopsis thaliana

Referring to FIG. 6, step 2 involves glutamate mutase (EC 5.4.99.1). Instep 2, the linear carbon chain of glutamate is rearranged to thebranched structure of threo-3-methylaspartate. This transformation iscatalyzed by glutamate mutase, a cobalamin-dependent enzyme composed oftwo subunits. Two glutamate mutases, from Clostridium cochlearium andClostridium tetanomorphum, have been cloned and functionally expressedin E. coli (Holloway and Marsh, J. Biol. Chem. 269:20425-20430 (1994);Reitzer et al., Acta Crystallogr. D Biol. Crystallogr. 54:1039-1042(1998)). The genes encoding this two-subunit protein are glmE and glmSfrom Clostridium cochlearium, mamA and glmE from Clostridiumtetanomorphum, and mutE and mutS from Clostridium tetani (Switzer,Glutamate mutase, pp. 289-305 Wiley, New York (1982)).

Gene GenBank ID GI Number Organism glmE P80077.2 2507035 Clostridiumcochlearium glmS P80078.2 17865765 Clostridium cochlearium mamA Q05488.1729588 Clostridium tetanomorphum glmE Q05509.1 729586 Clostridiumtetanomorphum mutE NP_783086.1 28212142 Clostridium tetani E88 mutSNP_783088.1 28212144 Clostridium tetani E88

Referring to FIG. 6, step 3 involves 3-methylaspartase (EC 4.3.1.2).3-methylaspartase, also referred to as beta-methylaspartase or3-methylaspartate ammonia-lyase, catalyzes the deamination ofthreo-3-methylasparatate to mesaconate. The 3-methylaspartase fromClostridium tetanomorphum has been cloned, functionally expressed in E.coli, and crystallized (Asuncion et al., Acta Crystallogr. D Biol.Crystallogr. 57:731-733 (2001); Asuncion et al., J. Biol. Chem.277:8306-8311 (2002); Botting et al., Biochemistry 27:2953-2955 (1988);Goda et al., Biochemistry 31:10747-10756 (1992)). In Citrobacteramalonaticus, this enzyme is encoded by BAA28709 (Kato and Asano, Arch.Microbiol. 168:457-463 (1997)). 3-methylaspartase has also beencrystallized from E. coli YG1002 (Asano and Kato, FEMS Microbiol. Lett.118:255-258 (1994)), although the protein sequence is not listed inpublic databases such as GenBank. Sequence homology can be used toidentify additional candidate genes, including CTC_(—)02563 in C. tetaniand ECs0761 in Escherichia coli O157:H7.

Gene GenBank ID GI Number Organism MAL AAB24070.1 259429 Clostridiumtetanomorphum BAA28709 BAA28709.1 3184397 Citrobacter amalonaticusCTC_02563 NP_783085.1 28212141 Clostridium tetani ECs0761 BAB34184.113360220 Escherichia coli O157:H7 str. Sakai

Referring to FIG. 6, step 4 involves mesaconate decarboxylase (EC4.1.1.-). The final step of the pathway entails the decarboxylation ofmesaconate to methacrylic acid. An enzyme catalyzing this exact reactionhas not been demonstrated experimentally. However, several enzymescatalyzing highly similar reactions exist. One enzyme with closelyrelated function is aconitate decarboxylase. This enzyme catalyzes thefinal step in itaconate biosynthesis in a strain of Candida and thefilamentous fungi Aspergillus terreus (Bonnarme et al., J. Bacteriol.177:3573-3578 (1995); Willke and Vorlop, Appl. Microbiol. Biotechnol.56:289-295 (2001)). Although itaconate is a compound of biotechnologicalinterest, no efforts have been made thus far to identify or clone theaconitate decarboxylase gene.

A second enzyme with similar function is 4-oxalocronate decarboxylase.This enzyme is common in a variety of organisms and the genes encodingthe enzyme from Pseudomonas sp. (strain 600) have been cloned andexpressed in E. coli (Shingler et al., J. Bacteriol. 174:711-724(1992)). The methyl group in mesaconate may cause steric hindrance, butthis problem could likely be overcome with directed evolution or proteinengineering. 4-oxalocronate decarboxylase is composed of two subunits.Genes encoding this enzyme include dmpH and dmpE in Pseudomonas sp.(strain 600) (Shingler et al., J. Bacteriol. 174:711-724 (1992)), xylIIand xylIII from Pseudomonas putida (Kato and Asano, Arch. Microbiol.168:457-463 (1997); Stanley et al., Biochemistry 39:718-726 (2000)), andReut_B5691 and Reut_B5692 from Ralstonia eutropha JMP134 (Hughes et al.,J. Bacteriol. 158:79-83 (1984)).

Gene GenBank ID GI Number Organism dmpH CAA43228.1 45685 Pseudomonas sp.CF600 dmpE CAA43225.1 45682 Pseudomonas sp. CF600 xylII YP_709328.1111116444 Pseudomonas putida xylIII YP_709353.1 111116469 Pseudomonasputida Reut_B5691 YP_299880.1 73539513 Ralstonia eutropha JMP134Reut_B5692 YP_299881.1 73539514 Ralstonia eutropha JMP134

This example describes a biosynthetic pathway for production of MMA fromalpha-ketoglutarate.

EXAMPLE IX Pathway for Conversion of Alpha-Ketoglutarate to MAA Via2-Hydroxyglutarate

This example describes an exemplary MAA synthesis pathway fromalph-ketoglutarate to MAA via 2-hydroxyglutarate.

Another exemplary pathway for MAA biosynthesis has a scheme similar tothe pathway described in Example VIII, but it passes through thehydroxylated intermediates 2-hydroxyglutarate and 3-methylmalate (seeFIG. 7), rather than amine-substituted intermediates (see FIG. 6).

Referring to FIG. 7, step 1 involves alpha-ketoglutarate reductase (EC1.1.99.2). The first step of this pathway entails the reduction ofalpha-ketoglutarate to 2-hydroxyglutarate by native enzymealpha-ketoglutarate reductase. This enzyme is encoded by serA, amultifunctional enzyme which also catalyzes the reduction of3-phosphoglycerate in central metabolism (Zhao and Winkler, J.Bacteriol. 178:232-239 (1996)). Genes L2HGDH in Homo sapiens (Jansen andWanders, Biochim. Biophys. Acta 1225:53-56 (1993)), FN0487 in L2hgdh inFusobacterium nucleatum (Hayashi et al., J. Nihon Univ. Sch. Dent.28:12-21 (1986)), and L2hgdh_predicted in Rattus norvegicus (Jansen andWanders, Biochim. Biophys. Acta 1225:53-56 (1993)) encode this enzyme.Gene candidates with high sequence homology include L2hgdh in Musmusculus and L2HGDH in Bos taurus. At high concentrations,2-hydroxyglutarate has been shown to feed back on alpha-ketoglutaratereductase activity by competitive inhibition (Zhao and Winkler, J.Bacteriol. 178:232-239. (1996)).

Gene GenBank ID GI Number Organism serA CAA01762.1 1247677 Escherichiacoli L2HGDH Q9H9P8.3 317373422 Homo sapiens L2hgdh NP_663418.1 21703884Mus musculus L2hgdh_predicted NP_001101498.1 157820173 Rattus norvegicusL2HGDH NP_001094560.1 155371911 Bos taurus FN0487 Q8RG31 81763568Fusobacterium nucleatum subsp. Nucleatum

Referring to FIG. 7, step 2 involves 2-hydroxyglutamate mutase (EC5.4.99.-). In the second step of the pathway, the carbon backboneundergoes rearrangement by a glutamate mutase enzyme. The most commonreaction catalyzed by such an enzyme is the conversion of glutamate tothreo-3-methylasparate, shown in step 2 of FIG. 6. Theadenosylcobalamin-dependent glutamate mutase from Clostridiumcochlearium has also been shown to react with 2-hydroxyglutarate as analternate substrate (Roymoulik et al., Biochemistry 39:10340-10346(2000)), although the rate of this reaction is two orders of magnitudelower with 2-hydroxyglutarate compared to the rate with native substrateglutamate. Directed evolution of the enzyme can be used to increaseglutamate mutase affinity for 2-hydroxyglutarate. GenBank accessionnumbers of protein sequences encoding glutamate mutases are found inExample VIII, step 2 of the pathway.

Referring to FIG. 7, step 3 involves 3-methylmalate dehydratase (EC4.2.1.-). In the third step, 3-methylmalate is dehydrated to formmesaconate. Although an enzyme catalyzing this exact transformation hasnot been described in the literature, several enzymes are able tocatalyze a similar reaction. One such enzyme is 2-methylmalatedehydratase, also called citramalate hydrolyase, which converts2-methylmalate to mesaconate. 2-Methylmalate and 3-methylmalate areclosely related, with the only difference in structure being thelocation of the hydroxyl group. 2-Methylmalate dehydratase activity wasdetected in Clostridium tetanomorphum, Morganella morganii, Citrobacteramalonaticus in the context of the glutamate degradation VI pathway(Kato and Asano, Arch. Microbiol. 168:457-463 (1997)); however the genesencoding this enzyme have not been sequenced to date.

A second candidate enzyme is fumarate hydratase, which catalyzes thedehydration of malate to fumarate. As described in Example V (step 5), awealth of structural information is available for this enzyme and otherstudies have successfully engineered the enzyme to alter activity,inhibition and localization (Weaver, Acta Crystallogr. D Biol.Crystallogr. 61:1395-1401 (2005)). Gene candidates are discussed inExample V, step 5 of the pathway.

Referring to FIG. 7, step 4 involves mesaconate decarboxylase (EC4.1.1.-). The final pathway step involves the decarboxylation ofmesaconate to methacrylic acid. This reaction is identical to the finalstep of the pathway described in Example VIII.

This example describes a biosynthetic pathway for production of MMA fromalpha-ketoglutarate.

EXAMPLE X Pathway for Conversion of Acetyl-CoA to 2-HydroxyisobutyricAcid or MAA

This example describes an exemplary 2-hydroxyisobutyric acid or MAAsynthesis pathway from acetyl-CoA.

MAA biosynthesis can proceed from acetyl-CoA in a minimum of fiveenzymatic steps (see FIG. 8). In this pathway, two molecules ofacetyl-CoA are combined to form acetoacetyl-coA, which is then reducedto 3-hydroxybutyryl-CoA. Alternatively, 4-hydroxybutyryl-CoA can beconverted to 3-hydroxybutyryl-CoA by way of 4-hydroxybutyryl-CoAdehydratase and crotonase (Martins et al., Proc. Nat. Acad. Sci. USA101(44) 15645-15649 (2004); Jones and Woods, Microbiol. Rev. 50:484-524(1986); Berg et al., Science 318(5857) 1782-1786 (2007)). A methylmutasethen rearranges the carbon backbone of 3-hydroxybutyryl-CoA to2-hydroxyisobutyryl-CoA, which is then dehydrated to formmethacrylyl-CoA. Alternatively, 2-hydroxyisobutyryl-CoA can be convertedto 2-hydroxyisobutyrate, secreted, and recovered as product. The finalstep converting methacrylyl-CoA to MAA can be performed by a singleenzyme (shown in FIG. 8) or a series of enzymes.

Referring to FIG. 8, step 1 involves acetoacetyl-CoA thiolase (EC2.3.1.9). The formation of acetoacetyl-CoA from two acetyl-CoA units iscatalyzed by acetyl-CoA thiolase. This enzyme is native to E. coli,encoded by gene atoB, and typically operates in theacetoacetate-degrading direction during fatty acid oxidation (Duncombeand Frerman, Arch. Biochem. Biophys. 176:159-170 (1976); Frerman andDuncombe, Biochim. Biophys. Acta 580:289-297 (1979)). The gene thlA fromClostridium acetobutylicum was engineered into an isopropanol-producingstrain of E. coli (Hanai et al., Appl. Environ. Microbiol. 73:7814-7818(2007); Stim-Herndon et al., Gene 154:81-85 (1995)). Additional genecandidates include thl from Clostridium pasteurianum (Meng and Li.Cloning, Biotechnol. Lett. 28:1227-1232 (2006)) and ERG10 from S.cerevisiae (Hiser et al, J Biol Chem 269:31383-89 (1994)).

Protein GenBank ID GI Number Organism atoB NP_416728 16130161Escherichia coli thlA NP_349476.1 15896127 Clostridium acetobutylicumthlB NP_149242.1 15004782 Clostridium acetobutylicum thl ABA18857.175315385 Clostridium pasteurianum ERG10 NP_015297 6325229 Saccharomycescerevisiae

Referring to FIG. 8, step 2 involves acetoacetyl-CoA reductase (EC#:1.1.1.35). The second step entails the reduction of acetoacetyl-CoA to3-hydroxybutyryl-CoA by acetoacetyl-CoA reductase. This enzymeparticipates in the acetyl-CoA fermentation pathway to butyrate inseveral species of Clostridia and has been studied in detail (Jones andWoods, Microbiol. Rev. 50:484-524 (1986)). The enzyme from Clostridiumacetobutylicum, encoded by hbd, has been cloned and functionallyexpressed in E. coli (Youngleson et al., J. Bacteriol. 171:6800-6807(1989)). Additionally, subunits of two fatty acid oxidation complexes inE. coli, encoded by fadB and fadJ, function as 3-hydroxyacyl-CoAdehydrogenases (Binstock and Schulz, Methods Enzymol. 71 Pt C:403-411(1981)). Yet other genes demonstrated to reduce acetoacetyl-CoA to3-hydroxybutyryl-CoA are phbB from Zoogloea ramigera (Ploux et al., Eur.J Biochem. 174:177-182 (1988)) and phaB from Rhodobacter sphaeroides(Alber et al., Mol. Microbiol 61:297-309 (2006)). The former gene isNADPH-dependent, its nucleotide sequence has been determined (Peoples etal., Mol. Microbiol 3:349-357 (1989)) and the gene has been expressed inE. coli. Substrate specificity studies on the gene led to the conclusionthat it could accept 3-oxopropionyl-CoA as a substrate besidesacetoacetyl-CoA (Ploux et al., Eur. J. Biochem. 174:177-182 (1988)).Additional gene candidates include Hbd1 (C-terminal domain) and Hbd2(N-terminal domain) in Clostridium kluyveri (Hillmer and Gottschalk,Biochim. Biophys. Acta 3334:12-23 (1974)) and HSD17B10 in Bos taurus(Wakil et al., J. Biol. Chem. 207:631-638 (1954)). The enzyme fromParacoccus denitrificans has been functionally expressed andcharacterized in E. coli (Yabutani et al., FEMS Microbiol Lett.133:85-90 (1995)). A number of similar enzymes have been found in otherspecies of Clostridia and in Metallosphaera sedula (Berg et al.,Science. 318:1782-1786 (2007)). The enzyme from Candida tropicalis is acomponent of the peroxisomal fatty acid beta-oxidation multifunctionalenzyme type 2 (MFE-2). The dehydrogenase B domain of this protein iscatalytically active on acetoacetyl-CoA. The domain has beenfunctionally expressed in E. coli, a crystal structure is available, andthe catalytic mechanism is well-understood (Ylianttila et al., BiochemBiophys Res Commun 324:25-30 (2004); Ylianttila et al., J Mol Biol358:1286-1295 (2006)).

Protein GENBANK ID GI NUMBER ORGANISM fadB P21177.2 119811 Escherichiacoli fadJ P77399.1 3334437 Escherichia coli Hbd2 EDK34807.1 146348271Clostridium kluyveri Hbd1 EDK32512.1 146345976 Clostridium kluyveriHSD17B10 O02691.3 3183024 Bos taurus phbB P23238.1 130017 Zoogloearamigera phaB YP_353825.1 77464321 Rhodobacter sphaeroides phaB BAA08358675524 Paracoccus denitrificans Hbd NP_349314.1 15895965 Clostridiumacetobutylicum Hbd AAM14586.1 20162442 Clostridium beijerinckiiMsed_1423 YP_001191505 146304189 Metallosphaera sedula Msed_0399YP_001190500 146303184 Metallosphaera sedula Msed_0389 YP_001190490146303174 Metallosphaera sedula Msed_1993 YP_001192057 146304741Metallosphaera sedula Fox2 Q02207 399508 Candida tropicalis

Referring to FIG. 8, step 3 involves 3-hydroxybutyryl-CoA mutase (EC5.4.99.-). In the next step, 3-hydroxybutyryl-CoA is rearranged to form2-hydroxyisobutyryl-CoA (2-HIBCoA) by 3-hydroxybutyryl-CoA mutase. Thisenzyme is a novel ICM-like methylmutase recently discovered andcharacterized in Methylibium petroleiphilum (Ratnatilleke et al., J.Biol. Chem. 274:31679-31685 (1999); Rohwerder et al., Appl. Environ.Microbiol. 72:4128-4135 (2006)). This enzyme, encoded by Mpe_B0541 inMethylibium petroleiphilum PM1, has high sequence homology to the largesubunit of methylmalonyl-CoA mutase in other organisms includingRsph17029_(—)3657 in Rhodobacter sphaeroides and Xaut_(—)5021 inXanthobacter autotrophicus. As discussed in Example VII (step 1),changes to a single amino acid near the active site alters the substratespecificity of the enzyme (Ratnatilleke et al., supra, 1999; Rohwerderet al., supra, 2006), so directed engineering of similar enzymes at thissite, such as methylmalonyl-CoA mutase or isobutryryl-CoA mutasedescribed previously, can be used to achieve the desired reactivity.

Gene GenBank ID GI Number Organism Mpe_B0541 YP_001023546.1 124263076Methylibium petroleiphilum PM1 Rsph17029_3657 YP_001045519.1 126464406Rhodobacter sphaeroides Xaut_5021 YP_001409455.1 154243882 Xanthobacterautotrophicus Py2

Referring to FIG. 8, step 4 involves 2-hydroxyisobutyryl-CoAdehydratase. The dehydration of 2-hydroxyacyl-CoA such as2-hydroxyisobutyryl-CoA can be catalyzed by a special class ofoxygen-sensitive enzymes that dehydrate 2-hydroxyacyl-CoA derivativesvia a radical-mechanism (Buckel and Golding, Annu. Rev. Microbiol.60:27-49 (2006); Buckel et al., Curr. Opin. Chem. Biol. 8:462-467(2004); Buckel et al., Biol. Chem. 386:951-959 (2005); Kim et al., FEBSJ. 272:550-561 (2005); Kim et al., FEMS Microbiol. Rev. 28:455-468(2004); Zhang et al., Microbiology 145 (Pt 9):2323-2334 (1999)). Oneexample of such an enzyme is the lactyl-CoA dehydratase from Clostridiumpropionicum, which catalyzes the dehydration of lactoyl-CoA to formacryl-CoA (Kuchta and Abeles, J. Biol. Chem. 260:13181-13189 (1985);Hofineister and Buckel, Eur. J. Biochem. 206:547-552 (1992)). Anadditional example is 2-hydroxyglutaryl-CoA dehydratase encoded byhgdABC from Acidaminococcus fermentans (Mueller and Buckel, Eur. J.Biochem. 230:698-704 (1995); Schweiger et al., Eur. J. Biochem.169:441-448 (1987)). Yet another example is the 2-hydroxyisocaproyl-CoAdehydratase from Clostridium difficile catalyzed by hadBC and activatedby hadI (Darley et al., FEBS J. 272:550-61 (2005)). The correspondingsequences for A. fermentans and C. difficile can be found as listedbelow. The sequence of the complete C. propionicium lactoyl-CoAdehydratase is not yet listed in publicly available databases. However,the sequence of the beta-subunit corresponds to the GenBank accessionnumber AJ276553 (Selmer et al, Eur J Biochem, 269:372-80 (2002)).

GenBank Gene Accession No. GI No. Organism hgdA P11569 296439332Acidaminococcus fermentans hgdB P11570 296439333 Acidaminococcusfermentans hgdC P11568 2506909 Acidaminococcus fermentans hadBYP_001086863 126697966 Clostridium difficile hadC YP_001086864 126697967Clostridium difficile hadI YP_001086862 126697965 Clostridium difficilelcdB AJ276553 7242547 Clostridium propionicum

Referring to FIG. 8, steps 5 or 6 involve a transferase (EC 2.8.3.-),hydrolase (EC 3.1.2.-), or synthetase (EC 6.2.1.-) with activity on amethacrylic acid or 2-hydroxyisobutyric acid, respectively. Directconversion of methacrylyl-CoA to MAA or 2-hydroxyisobutyryl-CoA to2-hydrioxyisobutyrate can be accomplished by a CoA transferase,synthetase or hydrolase. As discussed in Example VII, pathway energeticsare most favorable if a CoA transferase or a CoA synthetase is employedto accomplish this transformation. In the transferase family, the enzymeacyl-CoA:acetate-CoA transferase, also known as acetate-CoA transferase,is a suitable candidate to catalyze the desired 2-hydroxyisobutyryl-CoAor methacryl-CoA transferase activity due to its broad substratespecificity that includes branched acyl-CoA substrates (Matthies andSchink, Appl. Environ. Microbiol. 58:1435-1439 (1992); Vanderwinkel etal., Biochem. Biophys. Res. Commun. 33:902-908 (1968)). ADP-formingacetyl-CoA synthetase (ACD) is a promising enzyme in the CoA synthetasefamily operating on structurally similar branched chain compounds(Brasen and Schonheit, Arch. Microbiol. 182:277-287 (2004); Musfeldt andSchonheit, J. Bacteriol. 184:636-644 (2002)). In the CoA-hydrolasefamily, the enzyme 3-hydroxyisobutyryl-CoA hydrolase has been shown tooperate on a variety of branched chain acyl-CoA substrates including3-hydroxyisobutyryl-CoA, methylmalonyl-CoA, and3-hydroxy-2-methylbutanoyl-CoA (Hawes et al., Methods Enzymol.324:218-228 (2000); Hawes et al., J. Biol. Chem. 271:26430-26434 (1996);Shimomura et al., J. Biol. Chem. 269:14248-14253 (1994)). Additionalexemplary gene candidates for CoA transferases, synthetases, andhydrolases are discussed in Example VII (step 2 and 5).

Referring to FIG. 8, an alternative step 5 involves indirect conversionto MAA. As an alternative to direct conversion of MAA-CoA to MAA, analternate strategy for converting methacrylyl-CoA into MAA entails amulti-step process in which MAA-CoA is converted to MAA via3-hydroxyisobutyrate. By this process, MAA-CoA is first converted to3-hydroxyisobutyryl-CoA, which can subsequently be converted to MAA asdescribed in Example VII.

The first step of this indirect route entails the conversion of MAA-CoAto 3-hydroxyisobutyryl-CoA (3HIB-CoA) by enoyl-CoA hydratase (EC4.2.1.17 and 4.2.1.74). In E. coli, the gene products offadA and fadBencode a multienzyme complex involved in fatty acid oxidation thatexhibits enoyl-CoA hydratase activity (Nakahigashi and Inokuchi, NucleicAcids Research 18:4937 (1990); Yang, J. Bacteriol. 173:7405-7406 (1991);Yang et al., J. Biol. Chem. 265:10424-10429 (1990); Yang et al.,Biochemistry 30:6788-6795 (1991)). Knocking out a negative regulatorencoded by fadR can be utilized to activate the fadB gene product (Satoet al., J. Biosci. Bioengineer. 103:38-44 (2007)). The fadI and fadJgenes encode similar functions and are naturally expressed underanaerobic conditions (Campbell et al., Mol. Microbiol. 47:793-805(2003)).

GenBank Gene Accession No. GI No. Organism fadA YP_026272.1 49176430Escherichia coli fadB NP_418288.1 16131692 Escherichia coli fadINP_416844.1 16130275 Escherichia coli fadJ NP_416843.1 16130274Escherichia coli fadR NP_415705.1 16129150 Escherichia coli

Additional native gene candidates encoding an enoyl-CoA hydrataseinclude maoC (Park and Lee, J. Bacteriol. 185:5391-5397 (2003)), paaF(Ismail et al., Eur. J. Biochem. 270:3047-3054 (2003); Park and Lee,Appl. Biochem. Biotechnol. 113-116:335-346 (2004); Park and Yup,Biotechnol. Bioeng. 86:681-686. (2004)), and paaG (Ismail et al., Eur.J. Biochem. 270:3047-3054 (2003); Park and Lee, Appl. Biochem.Biotechnol. 113-116:335-346 (2004); Park and Yup, Biotechnol. Bioeng.86:681-686 (2004)). Other candidates include paaA, paaB, and paaN fromP. putida (Olivera et al., Proc. Natl. Acad. Sci. USA 95:6419-6424(1998)) and P. fluorescens (Di Gennaro et al., Arch. Microbiol.188:117-125 (2007)). The gene product of crt from C. acetobutylicum isanother candidate (Atsumi et al., Metab. Eng. epub Sep. 14, 2007;Boynton et al., J. Bacteriol. 178:3015-3024 (1996)). The enoyl-CoAhydratase of Pseudomonas putida, encoded by ech, catalyzes theconversion of 3-hydroxybutyryl-CoA to crotonyl-CoA (Roberts et al.,Arch. Microbiol 117:99-108 (1978)). This transformation is alsocatalyzed by the crt gene product of Clostridium acetobutylicum, thecrt1 gene product of C. kluyveri, and other clostridial organisms Atsumiet al., Metab Eng 10:305-311 (2008); Boynton et al., J Bacteriol.178:3015-3024 (1996); Hillmer et al., FEBS Lett. 21:351-354 (1972)).Additional enoyl-CoA hydratase candidates are phaA and phaB, of P.putida, and paaA and paaB from P. fluorescens (Olivera et al., Proc.Natl. Acad. Sci U.S.A 95:6419-6424 (1998)).

GenBank Gene Accession No. GI No. Organism maoC NP_415905.1 16129348Escherichia coli paaF NP_415911.1 16129354 Escherichia coli paaGNP_415912.1 16129355 Escherichia coli paaN NP_745413.1 26989988Pseudomonas putida (phaL) paaN ABF82246.1 106636106 Pseudomonasfluorescens ech NP_745498.1 26990073 Pseudomonas putida crt NP_349318.115895969 Clostridium acetobutylicum crt1 YP_001393856 153953091Clostridium kluyveri phaA NP_745427.1 26990002 Pseudomonas putida phaBNP_745426.1 26990001 Pseudomonas putida paaA ABF82233.1 106636093Pseudomonas fluorescens paaB ABF82234.1 106636094 Pseudomonasfluorescens

This example describes a biosynthetic pathway for production of2-hydroxyisobutyrate or MAA from acetyl-CoA.

EXAMPLE XI Pathway for Conversion of Acetyl-CoA to MAA Via Crotonoyl-CoA

This example describes an exemplary MAA synthetic pathway fromacetyl-CoA via crotonoyl-CoA.

Another route for converting acetyl-CoA to MAA in a minimum of sevenenzymatic steps is described (see FIG. 9). The yields of this pathwayunder aerobic and anaerobic conditions are similar to the pathwaydescribed in Example X.

The first two steps of the pathway are identical to steps 1 and 2 in thepathway described in Example X. In the third step, 3-HBCoA is dehydratedto form crotonyl-CoA by a crotonase (EC#: 4.2.1.55). The double bond incrotonyl-CoA is reduced by butyryl-CoA dehydrogenase (EC#: 1.3.99.2).Both of these enzymes, just like the acetoacetyl-CoA reductase, are apart of the acetyl-CoA fermentation pathway to butyrate in Clostridiaspecies (Jones and Woods, Microbiol. Rev. 50:484-524 (1986)). In thesubsequent step, butyryl-CoA is converted into isobutyryl-CoA byisobutyryl-CoA mutase (5.4.99.12), an enzyme that can reversibly convertbutyryl-CoA into isobutyryl-CoA. This enzyme has been cloned andsequenced from Streptomyces cinnamonensis, and the recombinant enzymehas been characterized in E. coli (Ratnatilleke et al., J. Biol. Chem.274:31679-31685 (1999)). The next step in the pathway entails theconversion of isobutyryl-CoA into methacrylyl-CoA via 2-methyl-acylCoAdehydrogenase (EC #: 1.3.99.12). This transformation towardsmethacrylyl-CoA has been observed in Streptomyces species, and theassociated enzyme has been isolated and expressed in E. coli (Younglesonet al., J. Bacteriol. 171:6800-6807 (1989)). In the final step,methacrylyl-CoA is converted to MAA by either a single enzyme or aseries of enzymes, as described in Example X (step 5).

This example describes a biosynthetic pathway for production of MAA fromacetyl-CoA.

EXAMPLE XII Pathway for Conversion of Acrylyl-CoA to MAA

This example describes an exemplary MAA synthesis pathway fromacrylyl-CoA.

High yields of MAA can be obtained through the acrylyl-CoA pathway (seeFIG. 10). This pathway requires the activation of lactate to lactoyl-CoAfollowed by five, or optionally six, more steps for the conversion ofthis activated CoA molecule into MAA. The MAA yield from glucose usingthis pathway is 1.28 mol/mol of glucose and oxygen uptake is requiredfor attaining these yields. In the absence of oxygen, the expected yielddecreases from 1.28 mol to 1.09 mol/mol glucose consumed. Both theaerobic and anaerobic pathways are energy limited at maximum MAA yieldand do not generate any ATP.

MAA biosynthesis through the acrylyl-CoA pathway first requires theconversion of pyruvate into lactate via lactate dehydrogenase (EC1.1.1.28), an enzyme native to E. coli and many other organisms. Thethree subsequent steps, converting lactate into propionyl-CoA, arecatalyzed by enzymes in pyruvate fermentation pathways in severalunrelated bacteria such as Clostridium propionicum and Megasphaeraelsdenii (MetaCyc). Lactate-CoA transferase (EC 2.8.3.1), also known aspropionate-CoA transferase, converts lactate into lactoyl-CoA and canuse both propionate and lactate as substrates. This enzyme has beenpurified and characterized (Schweiger et al., Eur. J. Biochem.169:441-448 (1987)). Lactoyl-CoA is dehydrated into acrylyl-CoA usinglactoyl-CoA dehydratase (EC 4.2.1.54), an enzyme that has been a subjectof numerous studies (Hofineister and Buckel, Eur. J. Biochem.206:547-552. (1992); Kuchta and Abeles, J. Biol. Chem. 260:13181-13189(1985)). Subsequently, acrylyl-CoA is reduced to propionyl-CoA using theacryloyl-CoA reductase (EC 1.3.2.2, formerly 1.3.99.3) (Hetzel et al.,Eur. J Biochem. 270:902-910 (2003); Kuchta and Abeles, supra, 1985).

Referring to FIG. 10, in step 5, propionyl-CoA is converted intoS-methylmalonyl-CoA by propionyl-CoA carboxylase (6.4.1.3).Propionyl-CoA carboxylase has been purified from rat liver (Browner etal., J. Biol. Chem. 264:12680-12685 (1989); Kraus et al., J. Biol. Chem.258:7245-7248 (1983)) and has been isolated and characterized from humanliver as well (Kalousek et al., J. Biol. Chem. 255:60-65 (1980)).Carboxylation of propionyl-CoA into succinyl-CoA via this enzyme hasbeen identified as one of the mechanisms of propionate metabolism in E.coli (Evans et al., Biochem. J. 291 (Pt 3):927-932 (1993)), but verylittle is known about the genetics of the pathway.

The final steps of the pathway entail conversion of methylmalonyl-CoAinto MAA (lumped reaction in FIG. 10). Enzymes catalyzing thesereactions are described in Example V.

This example describes a biosynthetic pathway for production of MAA frompyruvate.

EXAMPLE XIII Pathway for Conversion of 2-Ketoisovalerate to MAA

This example describes an exemplary MAA synthetic pathway from2-ketoisovalerate.

The pathway (see FIG. 11) exploits multiple steps of the valinedegradation route described in several organisms, including Bacillussubtilis, Arabidopsis thaliana, and several species of Pseuodomonas butnot known to be present in E. coli or in S. cerevisiae. In the firststep of the valine degradation pathway, valine is converted into2-ketoisovalerate by branched-chain amino acid aminotransferase (EC2.6.1.24), an enzyme also native to E. coli (Matthies and Schink, Appl.Environ. Microbiol. 58:1435-1439 (1992); Rudman and Meister, J. Biol.Chem. 200:591-604 (1953)). The subsequent conversion of2-ketoisovalerate into isobutyryl-CoA, catalyzed by a branched-chainketo-acid dehydrogenase complex (EC 1.2.1.25), is the committing stepfor MAA biosynthesis via this route. Next, isobutyryl-CoA is convertedto methacrylyl-CoA via isobutyryl-CoA dehydrogenase (EC 1.3.99.12).Details for this step are described in Example XI. The final step,conversion of MAA-CoA to MAA, is described in Example V.

This example describes a biosynthetic pathway for production of MMA from2-ketoisovalerate.

EXAMPLE XIV Pathway for Conversion of 4-Hydroxybutyryl-CoA to2-Hydroxyisobutyrate or MAA Via 2-Hydroxyisobutyryl-CoA

This example describes an exemplary 2-hydroxyisobutyrate or MAAsynthesis pathway proceeding from 4-hydroxybutyryl-CoA that passesthrough 2-hydroxyisobutyryl-CoA.

The pathway first entails the dehydration of 4-hydroxybutyryl-CoA tovinylacetyl-CoA which is subsequently isomerized to crotonoyl-CoA.Crotonyl-CoA is hydrated to form 3-hydroxybutyryl-CoA, which isrearranged into 2-hydroxyisobutyryl-CoA. The final step of the2-hydroxyisobutyrate pathway involves eliminating the CoA functionalgroup from 2-hydroxyisobutyryl-CoA. The final steps in MAA synthesisinvolve the dehydration of 2-hydroxyisobutyryl-CoA followed by theremoval of the CoA functional group from methacrylyl-CoA. Genecandidates for the first three pathway steps, steps 7, 8, and 9 of FIG.8, are described below. Gene candidates for steps 3, 4, 5, and 6 of FIG.8 are discussed in Example X.

Referring to FIG. 8, steps 7 and 8 are carried out by4-hydroxybutyryl-CoA dehydratase enzymes. The enzymes from bothClostridium aminobutyrium and C. kluyveri catalyze the reversibleconversion of 4-hydroxybutyryl-CoA to crotonyl-CoA and also possess anintrinsic vinylacetyl-CoA Δ-isomerase activity (Scherf and Buckel, Eur.J. Biochem. 215:421-429 (1993); Scherf et al., Arch. Microbiol.161:239-245 (1994)). Both native enzymes have been purified andcharacterized, including the N-terminal amino acid sequences (Scherf andBuckel, supra, 1993; Scherf et al., supra, 1994). The abfD genes from C.aminobutyrium and C. kluyveri match exactly with these N-terminal aminoacid sequences, thus are encoding the 4-hydroxybutyryl-CoAdehydratases/vinylacetyl-CoA Δ-isomerase. In addition, abfD fromPorphyromonas gingivalis ATCC 33277 is another exemplary4-hydroxybutyryl-CoA dehydratase that can be identified throughhomology. The abfD gene product from Porphyromonas gingivalis and theMsed_(—)1220 gene product from Metallosphaera sedula are closely relatedby sequence homology to the Clostridial gene products.

GenBank Gene Accession No. GI No. Organism abfD YP_001396399.1 153955634Clostridium kluyveri DSM 555 abfD P55792 84028213 Clostridiumaminobutyricum abfD YP_001928843 188994591 Porphyromonas gingivalis(ATCC 33277) Msed_1220 YP_001191305.1 146303989 Metallosphaera sedula

Step 9 of FIG. 8 is carried out by a crotonase enzyme. Such enzymes arerequired for n-butanol formation in some organisms, particularlyClostridial species, and also comprise one step of the3-hydroxypropionate/4-hydroxybutyrate cycle in thermoacidophilic Archaeaof the genera Sulfolobus, Acidianus, and Metallosphaera. Exemplary genesencoding crotonase enzymes can be found in C. acetobutylicum (Boynton,et al., J. Bacteriol. 178(11):3015-3024 (1996)), C. kluyveri (Hillmerand Gottschalk, FEBS Lett. 21(3):351-354 (1972)), and Metallosphaerasedula (Berg et al., Science 318(5857):1782-1786 (2007)) though thesequence of the latter gene is not known. Enoyl-CoA hydratases, whichare involved in fatty acid beta-oxidation and/or the metabolism ofvarious amino acids, can also catalyze the hydration of crotonyl-CoA toform 3-hydroxybutyryl-CoA (Agnihotri and Liu, Bioorg. Med. Chem.11(1):9-20 (2003); Roberts et al., Arch. Microbiol. 117(1):99-108(1978); Conrad et al., J. Bacteriol. 118(1); 103-11 (1974)). Theenoyl-CoA hydratases, phaA and phaB, of P. putida are believed to carryout the hydroxylation of double bonds during phenylacetate catabolism(Olivera et al., Proc Natl Acad Sci USA 95(11):6419-6424 (1998)). ThepaaA and paaB from P. fluorescens catalyze analogous transformations(Olivera et al., supra, 1998). Lastly, a number of Escherichia coligenes have been shown to demonstrate enoyl-CoA hydratase functionalityincluding maoC (Park and Lee, J. Bacteriol. 185(18):5391-5397 (2003)),paaF (Park and Lee, Biotechnol. Bioeng. 86(6):681-686 (2004a)); Park andLee, Appl. Biochem. Biotechnol. 113-116: 335-346 (2004b)); Ismail et al.Eur. J. Biochem. 270(14):3047-3054 (2003), and paaG (Park and Lee,supra, 2004; Park and Lee, supra, 2004b; Ismail et al., supra, 2003).

GenBank Gene Accession No. GI No. Organism crt NP_349318.1 15895969Clostridium acetobutylicum crt1 YP_001393856 153953091 Clostridiumkluyveri DSM 555 phaA NP_745427.1 26990002 Pseudomonas putida phaBNP_745426.1 26990001 Pseudomonas putida paaA ABF82233.1 106636093Pseudomonas fluorescens paaB ABF82234.1 106636094 Pseudomonasfluorescens maoC NP_415905.1 16129348 Escherichia coli paaF NP_415911.116129354 Escherichia coli paaG NP_415912.1 16129355 Escherichia coli

This example describes a biosynthesis pathway for 2-hydroxyisobutyrateor methacylic acid from 4-hydroxybutyryl-CoA.

EXAMPLE XV Exemplary Hydrogenase and CO Dehydrogenase Enzymes forExtracting Reducing Equivalents from Syngas and Exemplary Reductive TCACycle Enzymes

Enzymes of the reductive TCA cycle useful in the non-naturally occurringmicrobial organisms of the present invention include one or more ofATP-citrate lyase and three CO₂-fixing enzymes: isocitratedehydrogenase, alpha-ketoglutarate:ferredoxin oxidoreductase,pyruvate:ferredoxin oxidoreductase. The presence of ATP-citrate lyase orcitrate lyase and alpha-ketoglutarate:ferredoxin oxidoreductaseindicates the presence of an active reductive TCA cycle in an organism.Enzymes for each step of the reductive TCA cycle are shown below anddescribed herein.

ATP-citrate lyase (ACL, EC 2.3.3.8), also called ATP citrate synthase,catalyzes the ATP-dependent cleavage of citrate to oxaloacetate andacetyl-CoA. ACL is an enzyme of the RTCA cycle that has been studied ingreen sulfur bacteria Chlorobium limicola and Chlorobium tepidum. Thealpha(4)beta(4) heteromeric enzyme from Chlorobium limicola was clonedand characterized in E. coli (Kanao et al., Eur. J. Biochem.269:3409-3416 (2002). The C. limicola enzyme, encoded by aclAB, isirreversible and activity of the enzyme is regulated by the ratio ofADP/ATP. A recombinant ACL from Chlorobium tepidum was also expressed inE. coli and the holoenzyme was reconstituted in vitro, in a studyelucidating the role of the alpha and beta subunits in the catalyticmechanism (Kim and Tabita, J. Bacteriol. 188:6544-6552 (2006). ACLenzymes have also been identified in Balnearium lithotrophicum,Sulfurihydrogenibium subterraneum and other members of the bacterialphylum Aquificae (Hugler et al., Environ. Microbiol. 9:81-92 (2007)).This activity has been reported in some fungi as well. Exemplaryorganisms include Sordaria macrospora (Nowrousian et al., Curr. Genet.37:189-93 (2000), Aspergillus nidulans, Yarrowia lipolytica (Hynes andMurray, Eukaryotic Cell, July: 1039-1048, (2010) and Aspergillus niger(Meijer et al. J. Ind. Microbiol. Biotechnol. 36:1275-1280 (2009). Othercandidates can be found based on sequence homology. Information relatedto these enzymes is tabulated below:

Protein GenBank ID GI Number Organism aclA BAB21376.1 12407237Chlorobium limicola aclB BAB21375.1 12407235 Chlorobium limicola aclAAAM72321.1 21647054 Chlorobium tepidum aclB AAM72322.1 21647055Chlorobium tepidum aclA ABI50076.1 114054981 Balnearium lithotrophicumaclB ABI50075.1 114054980 Balnearium lithotrophicum aclA ABI50085.1114055040 Sulfurihydrogenibium subterraneum aclB ABI50084.1 114055039Sulfurihydrogenibium subterraneum aclA AAX76834.1 62199504 Sulfurimonasdenitrificans aclB AAX76835.1 62199506 Sulfurimonas denitrificans acl1XP_504787.1 50554757 Yarrowia lipolytica acl2 XP_503231.1 50551515Yarrowia lipolytica SPBC1703.07 NP_596202.1 19112994 Schizosaccharomycespombe SPAC22A12.16 NP_593246.1 19114158 Schizosaccharomyces pombe acl1CAB76165.1 7160185 Sordaria macrospora acl2 CAB76164.1 7160184 Sordariamacrospora aclA CBF86850.1 259487849 Aspergillus nidulans aclB CBF86848259487848 Aspergillus nidulans

In some organisms the conversion of citrate to oxaloacetate andacetyl-CoA proceeds through a citryl-CoA intermediate and is catalyzedby two separate enzymes, citryl-CoA synthetase (EC 6.2.1.18) andcitryl-CoA lyase (EC 4.1.3.34) (Aoshima, M., Appl. Microbiol.Biotechnol. 75:249-255 (2007). Citryl-CoA synthetase catalyzes theactivation of citrate to citryl-CoA. The Hydrogenobacter thermophilusenzyme is composed of large and small subunits encoded by ccsA and ccsB,respectively (Aoshima et al., Mol. Micrbiol. 52:751-761 (2004)). Thecitryl-CoA synthetase of Aquifex aeolicus is composed of alpha and betasubunits encoded by sucC1 and sucD1 (Hugler et al., Environ. Microbiol.9:81-92 (2007)). Citryl-CoA lyase splits citryl-CoA into oxaloacetateand acetyl-CoA. This enzyme is a homotrimer encoded by ccl inHydrogenobacter thermophilus (Aoshima et al., Mol. Microbiol. 52:763-770(2004)) and aq_(—)150 in Aquifex aeolicus (Hugler et al., supra (2007)).The genes for this mechanism of converting citrate to oxaloacetate andcitryl-CoA have also been reported recently in Chlorobium tepidum (Eisenet al., PNAS 99(14): 9509-14 (2002).

Protein GenBank ID GI Number Organism ccsA BAD17844.1 46849514Hydrogenobacter thermophilus ccsB BAD17846.1 46849517 Hydrogenobacterthermophilus sucC1 AAC07285 2983723 Aquifex aeolicus sucD1 AAC076862984152 Aquifex aeolicus ccl BAD17841.1 46849510 Hydrogenobacterthermophilus aq_150 AAC06486 2982866 Aquifex aeolicus CT0380 NP_66128421673219 Chlorobium tepidum CT0269 NP_661173.1 21673108 Chlorobiumtepidum CT1834 AAM73055.1 21647851 Chlorobium tepidum

Oxaloacetate is converted into malate by malate dehydrogenase (EC1.1.1.37), an enzyme which functions in both the forward and reversedirection. S. cerevisiae possesses three copies of malate dehydrogenase,MDH1 (McAlister-Henn and Thompson, J. Bacteriol. 169:5157-5166 (1987),MDH2 (Minard and McAlister-Henn, Mol. Cell. Biol. 11:370-380 (1991);Gibson and McAlister-Henn, J. Biol. Chem. 278:25628-25636 (2003)), andMDH3 (Steffan and McAlister-Henn, J. Biol. Chem. 267:24708-24715(1992)), which localize to the mitochondrion, cytosol, and peroxisome,respectively. E. coli is known to have an active malate dehydrogenaseencoded by mdh.

Protein GenBank ID GI Number Organism MDH1 NP_012838 6322765Saccharomyces cerevisiae MDH2 NP_014515 116006499 Saccharomycescerevisiae MDH3 NP_010205 6320125 Saccharomyces cerevisiae MdhNP_417703.1 16131126 Escherichia coli

Fumarate hydratase (EC 4.2.1.2) catalyzes the reversible hydration offumarate to malate. The three fumarases of E. coli, encoded by fumA,fumB and fumC, are regulated under different conditions of oxygenavailability. FumB is oxygen sensitive and is active under anaerobicconditions. FumA is active under microanaerobic conditions, and FumC isactive under aerobic growth conditions (Tseng et al., J. Bacteriol.183:461-467 (2001); Woods et al., Biochim. Biophys. Acta 954:14-26(1988); Guest et al., J. Gen. Microbiol. 131:2971-2984 (1985)). S.cerevisiae contains one copy of a fumarase-encoding gene, FUM1, whoseproduct localizes to both the cytosol and mitochondrion (Sass et al., J.Biol. Chem. 278:45109-45116 (2003)). Additional fumarase enzymes arefound in Campylobacter jejuni (Smith et al., Int. J. Biochem. Cell.Biol. 31:961-975 (1999)), Thermus thermophilus (Mizobata et al., Arch.Biochem. Biophys. 355:49-55 (1998)) and Rattus norvegicus (Kobayashi etal., J. Biochem. 89:1923-1931 (1981)). Similar enzymes with highsequence homology include fum1 from Arabidopsis thaliana and fumC fromCorynebacterium glutamicum. The MmcBC fumarase from Pelotomaculumthermopropionicum is another class of fumarase with two subunits(Shimoyama et al., FEMS Microbiol. Lett. 270:207-213 (2007)).

Protein GenBank ID GI Number Organism fumA NP_416129.1 16129570Escherichia coli fumB NP_418546.1 16131948 Escherichia coli fumCNP_416128.1 16129569 Escherichia coli FUM1 NP_015061 6324993Saccharomyces cerevisiae fumC Q8NRN8.1 39931596 Corynebacteriumglutamicum fumC O69294.1 9789756 Campylobacter jejuni fumC P8412775427690 Thermus thermophilus

Protein GenBank ID GI Number Organism fumH P14408.1 120605 Rattusnorvegicus MmcB YP_001211906 147677691 Pelotomaculum thermopropionicumMmcC YP_001211907 147677692 Pelotomaculum thermopropionicum

Fumarate reductase catalyzes the reduction of fumarate to succinate. Thefumarate reductase of E. coli, composed of four subunits encoded byfrdABCD, is membrane-bound and active under anaerobic conditions. Theelectron donor for this reaction is menaquinone and the two protonsproduced in this reaction do not contribute to the proton gradient(Iverson et al., Science 284:1961-1966 (1999)). The yeast genome encodestwo soluble fumarate reductase isozymes encoded by FRDS1 (Enomoto etal., DNA Res. 3:263-267 (1996)) and FRDS2 (Muratsubaki et al., Arch.Biochem. Biophys. 352:175-181 (1998)), which localize to the cytosol andpromitochondrion, respectively, and are used during anaerobic growth onglucose (Arikawa et al., FEMS Microbiol. Lett. 165:111-116 (1998)).

Protein GenBank ID GI Number Organism FRDS1 P32614 418423 Saccharomycescerevisiae FRDS2 NP_012585 6322511 Saccharomyces cerevisiae frdANP_418578.1 16131979 Escherichia coli frdB NP_418577.1 16131978Escherichia coli frdC NP_418576.1 16131977 Escherichia coli frdDNP_418475.1 16131877 Escherichia coli

The ATP-dependent acylation of succinate to succinyl-CoA is catalyzed bysuccinyl-CoA synthetase (EC 6.2.1.5). The product of the LS' and LSC2genes of S. cerevisiae and the sucC and sucD genes of E. coli naturallyform a succinyl-CoA synthetase complex that catalyzes the formation ofsuccinyl-CoA from succinate with the concomitant consumption of one ATP,a reaction which is reversible in vivo (Buck et al., Biochemistry24:6245-6252 (1985)). These proteins are identified below:

Protein GenBank ID GI Number Organism LSC1 NP_014785 6324716Saccharomyces cerevisiae LSC2 NP_011760 6321683 Saccharomyces cerevisiaesucC NP_415256.1 16128703 Escherichia coli sucD AAC73823.1 1786949Escherichia coli

Alpha-ketoglutarate:ferredoxin oxidoreductase (EC 1.2.7.3), also knownas 2-oxoglutarate synthase or 2-oxoglutarate:ferredoxin oxidoreductase(OFOR), forms alpha-ketoglutarate from CO2 and succinyl-CoA withconcurrent consumption of two reduced ferredoxin equivalents. OFOR andpyruvate:ferredoxin oxidoreductase (PFOR) are members of a diversefamily of 2-oxoacid:ferredoxin (flavodoxin) oxidoreductases whichutilize thiamine pyrophosphate, CoA and iron-sulfur clusters ascofactors and ferredoxin, flavodoxin and FAD as electron carriers (Adamset al., Archaea. Adv. Protein Chem. 48:101-180 (1996)). Enzymes in thisclass are reversible and function in the carboxylation direction inorganisms that fix carbon by the RTCA cycle such as Hydrogenobacterthermophilus, Desulfobacter hydrogenophilus and Chlorobium species(Shiba et al. 1985; Evans et al., Proc. Natl. Acad. ScI. U.S.A. 55:92934(1966); Buchanan, 1971). The two-subunit enzyme from H. thermophilus,encoded by korAB, has been cloned and expressed in E. coli (Yun et al.,Biochem. Biophys. Res. Commun. 282:589-594 (2001)). A five subunit OFORfrom the same organism with strict substrate specificity forsuccinyl-CoA, encoded by forDABGE, was recently identified and expressedin E. coli (Yun et al. 2002). The kinetics of CO2 fixation of both H.thermophilus OFOR enzymes have been characterized (Yamamoto et al.,Extremophiles 14:79-85 (2010)). A CO2-fixing OFOR from Chlorobiumthiosulfatophilum has been purified and characterized but the genesencoding this enzyme have not been identified to date. Enzyme candidatesin Chlorobium species can be inferred by sequence similarity to the H.thermophilus genes. For example, the Chlorobium limicola genome encodestwo similar proteins. Acetogenic bacteria such as Moorella thermoaceticaare predicted to encode two OFOR enzymes. The enzyme encoded byMoth_(—)0034 is predicted to function in the CO2-assimilating direction.The genes associated with this enzyme, Moth_(—)0034 have not beenexperimentally validated to date but can be inferred by sequencesimilarity to known OFOR enzymes.

OFOR enzymes that function in the decarboxylation direction underphysiological conditions can also catalyze the reverse reaction. TheOFOR from the thermoacidophilic archaeon Sulfolobus sp. strain 7,encoded by ST2300, has been extensively studied (Zhang et al. 1996. Aplasmid-based expression system has been developed for efficientlyexpressing this protein in E. coli (Fukuda et al., Eur. J. Biochem.268:5639-5646 (2001)) and residues involved in substrate specificitywere determined (Fukuda and Wakagi, Biochim. Biophys. Acta 1597:74-80(2002)). The OFOR encoded by Ape1472/Ape1473 from Aeropyrum pernix str.K1 was recently cloned into E. coli, characterized, and found to reactwith 2-oxoglutarate and a broad range of 2-oxoacids (Nishizawa et al.,FEBS Lett. 579:2319-2322 (2005)). Another exemplary OFOR is encoded byoorDABC in Helicobacter pylori (Hughes et al. 1998). An enzyme specificto alpha-ketoglutarate has been reported in Thauera aromatics (Dornerand Boll, J, Bacteriol. 184 (14), 3975-83 (2002). A similar enzyme canbe found in Rhodospirillum rubrum by sequence homology. A two subunitenzyme has also been identified in Chlorobium tepidum (Eisen et al.,PNAS 99(14): 9509-14 (2002)).

Protein GenBank ID GI Number Organism korA BAB21494 12583691Hydrogenobacter thermophilus korB BAB21495 12583692 Hydrogenobacterthermophilus forD BAB62132.1 14970994 Hydrogenobacter thermophilus forABAB62133.1 14970995 Hydrogenobacter thermophilus forB BAB62134.114970996 Hydrogenobacter thermophilus forG BAB62135.1 14970997Hydrogenobacter thermophilus forE BAB62136.1 14970998 Hydrogenobacterthermophilus Clim_0204 ACD89303.1 189339900 Chlorobium limicolaClim_0205 ACD89302.1 189339899 Chlorobium limicola Clim_1123 ACD90192.1189340789 Chlorobium limicola Clim_1124 ACD90193.1 189340790 Chlorobiumlimicola Moth_1984 YP_430825.1 83590816 Moorella thermoacetica Moth_1985YP_430826.1 83590817 Moorella thermoacetica Moth_0034 YP_428917.183588908 Moorella thermoacetica ST2300 NP_378302.1 15922633 Sulfolobussp. strain 7 Ape1472 BAA80470.1 5105156 Aeropyrum pernix Ape1473BAA80471.2 116062794 Aeropyrum pernix oorD NP_207383.1 15645213Helicobacter pylori oorA NP_207384.1 15645214 Helicobacter pylori oorBNP_207385.1 15645215 Helicobacter pylori oorC NP_207386.1 15645216Helicobacter pylori CT0163 NP_661069.1 21673004 Chlorobium tepidumCT0162 NP_661068.1 21673003 Chlorobium tepidum korA CAA12243.2 19571179Thauera aromatica korB CAD27440.1 19571178 Thauera aromatica Rru_A2721YP_427805.1 83594053 Rhodospirillum rubrum Rru_A2722 YP_427806.183594054 Rhodospirillum rubrum

Isocitrate dehydrogenase catalyzes the reversible decarboxylation ofisocitrate to 2-oxoglutarate coupled to the reduction of NAD(P)⁺. IDHenzymes in Saccharomyces cerevisiae and Escherichia coli are encoded byIDP1 and icd, respectively (Haselbeck and McAlister-Henn, J. Biol. Chem.266:2339-2345 (1991); Nimmo, H. G., Biochem. J. 234:317-2332 (1986)).The reverse reaction in the reductive TCA cycle, the reductivecarboxylation of 2-oxoglutarate to isocitrate, is favored by theNADPH-dependent CO₂-fixing IDH from Chlorobium limicola and wasfunctionally expressed in E. coli (Kanao et al., Eur. J. Biochem.269:1926-1931 (2002)). A similar enzyme with 95% sequence identity isfound in the C. tepidum genome in addition to some other candidateslisted below.

Protein GenBank ID GI Number Organism Icd ACI84720.1 209772816Escherichia coli IDP1 AAA34703.1 171749 Saccharomyces cerevisiae IdhBAC00856.1 21396513 Chlorobium limicola Icd AAM71597.1 21646271Chlorobium tepidum icd NP_952516.1 39996565 Geobacter sulfurreducens icdYP_393560. 78777245 Sulfurimonas denitrificans

In H. thermophilus the reductive carboxylation of 2-oxoglutarate toisocitrate is catalyzed by two enzymes: 2-oxoglutarate carboxylase andoxalosuccinate reductase. 2-Oxoglutarate carboxylase (EC 6.4.1.7)catalyzes the ATP-dependent carboxylation of alpha-ketoglutarate tooxalosuccinate (Aoshima and Igarashi, Mol. Microbiol. 62:748-759(2006)). This enzyme is a large complex composed of two subunits.Biotinylation of the large (A) subunit is required for enzyme function(Aoshima et al., Mol. Microbiol. 51:791-798 (2004)). Oxalosuccinatereductase (EC 1.1.1.-) catalyzes the NAD-dependent conversion ofoxalosuccinate to D-threo-isocitrate. The enzyme is a homodimer encodedby icd in H. thermophilus. The kinetic parameters of this enzymeindicate that the enzyme only operates in the reductive carboxylationdirection in vivo, in contrast to isocitrate dehydrogenase enzymes inother organisms (Aoshima and Igarashi, J. Bacteriol. 190:2050-2055(2008)). Based on sequence homology, gene candidates have also beenfound in Thiobacillus denitrificans and Thermocrinis albus.

Protein GenBank ID GI Number Organism cfiA BAF34932.1 116234991Hydrogenobacter thermophilus cifB BAF34931.1 116234990 Hydrogenobacterthermophilus Icd BAD02487.1 38602676 Hydrogenobacter thermophilusTbd_1556 YP_315314 74317574 Thiobacillus denitrificans Tbd_1555YP_315313 74317573 Thiobacillus denitrificans Tbd_0854 YP_31461274316872 Thiobacillus denitrificans Thal_0268 YP_003473030 289548042Thermocrinis albus Thal_0267 YP_003473029 289548041 Thermocrinis albusThal_0646 YP_003473406 289548418 Thermocrinis albus

Aconitase (EC 4.2.1.3) is an iron-sulfur-containing protein catalyzingthe reversible isomerization of citrate and iso-citrate via theintermediate cis-aconitate. Two aconitase enzymes are encoded in the E.coli genome by acnA and acnB. AcnB is the main catabolic enzyme, whileAcnA is more stable and appears to be active under conditions ofoxidative or acid stress (Cunningham et al., Microbiology 143 (Pt12):3795-3805 (1997)). Two isozymes of aconitase in Salmonellatyphimurium are encoded by acnA and acnB (Horswill andEscalante-Semerena, Biochemistry 40:4703-4713 (2001)). The S. cerevisiaeaconitase, encoded by ACO1, is localized to the mitochondria where itparticipates in the TCA cycle (Gangloff et al., Mol. Cell. Biol.10:3551-3561 (1990)) and the cytosol where it participates in theglyoxylate shunt (Regev-Rudzki et al., Mol. Biol. Cell. 16:4163-4171(2005)).

Protein GenBank ID GI Number Organism acnA AAC7438.1 1787531 Escherichiacoli acnB AAC73229.1 2367097 Escherichia coli acnA NP_460671.1 16765056Salmonella typhimurium acnB NP_459163.1 16763548 Salmonella typhimuriumACO1 AAA34389.1 170982 Saccharomyces cerevisiae HP0779 NP_207572.115645398 Helicobacter pylori 26695 H16_B0568 CAJ95365.1 113529018Ralstonia eutropha DesfrDRAFT_3783 ZP_07335307.1 303249064 Desulfovibriofructosovorans JJ Suden_1040 ABB44318.1 78497778 Sulfurimonasdenitrificans (acnB) Hydth_0755 ADO45152.1 308751669 Hydrogenobacterthermophilus CT0543 (acn) AAM71785.1 21646475 Chlorobium tepidumClim_2436 YP_001944436.1 189347907 Chlorobium limicola Clim_0515ACD89607.1 189340204 Chlorobium limicola

Pyruvate:ferredoxin oxidoreductase (PFOR) catalyzes the reversibleoxidation of pyruvate to form acetyl-CoA. The PFOR from Desulfovibrioafricanus has been cloned and expressed in E. coli resulting in anactive recombinant enzyme that was stable for several days in thepresence of oxygen (Pieulle et al., J. Bacteriol. 179:5684-5692 (1997)).Oxygen stability is relatively uncommon in PFORs and is believed to beconferred by a 60 residue extension in the polypeptide chain of the D.africanus enzyme. Two cysteine residues in this enzyme form a disulfidebond that protects it against inactivation in the form of oxygen. Thisdisulfide bond and the stability in the presence of oxygen has beenfound in other Desulfovibrio species also (Vita et al., Biochemistry,47: 957-64 (2008)). The M. thermoacetica PFOR is also well characterized(Menon and Ragsdale, Biochemistry 36:8484-8494 (1997)) and was shown tohave high activity in the direction of pyruvate synthesis duringautotrophic growth (Furdui and Ragsdale, J. Biol. Chem. 275:28494-28499(2000)). Further, E. coli possesses an uncharacterized open readingframe, ydbK, encoding a protein that is 51% identical to the M.thermoacetica PFOR. Evidence for pyruvate oxidoreductase activity in E.coli has been described (Blaschkowski et al., Eur. J. Biochem.123:563-569 (1982)). PFORs have also been described in other organisms,including Rhodobacter capsulatas (Yakunin and Hallenbeck, Biochimica etBiophysica Acta 1409 (1998) 39-49 (1998)) and Choloboum tepidum (Eisenet al., PNAS 99(14): 9509-14 (2002)). The five subunit PFOR from H.thermophilus, encoded by porEDABG, was cloned into E. coli and shown tofunction in both the decarboxylating and CO₂-assimilating directions(Ikeda et al. 2006; Yamamoto et al., Extremophiles 14:79-85 (2010)).Homologs also exist in C. carboxidivorans P7. Several additional PFORenzymes are described in the following review (Ragsdale, S. W., Chem.Rev. 103:2333-2346 (2003)). Finally, flavodoxin reductases (e.g., fqrBfrom Helicobacter pylori or Campylobacter jejuni) (St Maurice et al., J.Bacteriol. 189:4764-4773 (2007)) or Rnf-type proteins (Seedorf et al.,Proc. Natl. Acad. Sci. U.S.A. 105:2128-2133 (2008); and Herrmann, J.Bacteriol 190:784-791 (2008)) provide a means to generate NADH or NADPHfrom the reduced ferredoxin generated by PFOR. These proteins areidentified below.

Protein GenBank ID GI Number Organism DesfrDRAFT_0121 ZP_07331646.1303245362 Desulfovibrio fructosovorans JJ Por CAA70873.1 1770208Desulfovibrio africanus por YP_012236.1 46581428 Desulfovibrio vulgarisstr. Hildenborough Dde_3237 ABB40031.1 78220682 DesulfoVibriodesulfuricans G20 Ddes_0298 YP_002478891.1 220903579 Desulfovibriodesulfuricans subsp. desulfuricans str. ATCC27774 Por YP_428946.183588937 Moorella thermoacetica YdbK NP_415896.1 16129339 Escherichiacoli nifJ (CT1628) NP_662511.1 21674446 Chlorobium tepidum CJE1649YP_179630.1 57238499 Campylobacter jejuni nifJ ADE85473.1 294476085Rhodobacter capsulatus porE BAA95603.1 7768912 Hydrogenobacterthermophilus porD BAA95604.1 7768913 Hydrogenobacter thermophilus porABAA95605.1 7768914 Hydrogenobacter thermophilus porB BAA95606.1 776891Hydrogenobacter thermophilus porG BAA95607.1 7768916 Hydrogenobacterthermophilus FqrB YP_001482096.1 157414840 Campylobacter jejuni HP1164NP_207955.1 15645778 Helicobacter pylori RnfC EDK33306.1 146346770Clostridium kluyveri RnfD EDK33307.1 146346771 Clostridium kluyveri RnfGEDK33308.1 146346772 Clostridium kluyveri RnfE EDK33309.1 146346773Clostridium kluyveri RnfA EDK33310.1 146346774 Clostridium kluyveri RnfBEDK33311.1 146346775 Clostridium kluyveri

The conversion of pyruvate into acetyl-CoA can be catalyzed by severalother enzymes or their combinations thereof. For example, pyruvatedehydrogenase can transform pyruvate into acetyl-CoA with theconcomitant reduction of a molecule of NAD into NADH. It is amulti-enzyme complex that catalyzes a series of partial reactions whichresults in acylating oxidative decarboxylation of pyruvate. The enzymecomprises of three subunits: the pyruvate decarboxylase (E1),dihydrolipoamide acyltransferase (E2) and dihydrolipoamide dehydrogenase(E3). This enzyme is naturally present in several organisms, includingE. coli and S. cerevisiae. In the E. coli enzyme, specific residues inthe E1 component are responsible for substrate specificity (Bisswanger,H., J. Biol. Chem. 256:815-82 (1981); Bremer, J., Eur. J. Biochem.8:535-540 (1969); Gong et al., J. Biol. Chem. 275:13645-13653 (2000)).Enzyme engineering efforts have improved the E. coli PDH enzyme activityunder anaerobic conditions (Kim et al., J. Bacteriol. 190:3851-3858(2008); Kim et al., Appl. Environ. Microbiol. 73:1766-1771 (2007); Zhouet al., Biotechnol. Lett. 30:335-342 (2008)). In contrast to the E. coliPDH, the B. subtilis complex is active and required for growth underanaerobic conditions (Nakano et al., J. Bacteriol. 179:6749-6755(1997)). The Klebsiella pneumoniae PDH, characterized during growth onglycerol, is also active under anaerobic conditions (5). Crystalstructures of the enzyme complex from bovine kidney (18) and the E2catalytic domain from Azotobacter vinelandii are available (4). Yetanother enzyme that can catalyze this conversion is pyruvate formatelyase. This enzyme catalyzes the conversion of pyruvate and CoA intoacetyl-CoA and formate. Pyruvate formate lyase is a common enzyme inprokaryotic organisms that is used to help modulate anaerobic redoxbalance. Exemplary enzymes can be found in Escherichia coli encoded bypflB (Knappe and Sawers, FEMS. Microbiol Rev. 6:383-398 (1990)),Lactococcus lactis (Melchiorsen et al., Appl Microbiol Biotechnol58:338-344 (2002)), and Streptococcus mutans (Takahashi-Abbe et al.,Oral. Microbiol Immunol. 18:293-297 (2003)). E. coli possesses anadditional pyruvate formate lyase, encoded by tdcE, that catalyzes theconversion of pyruvate or 2-oxobutanoate to acetyl-CoA or propionyl-CoA,respectively (Hesslinger et al., Mol. Microbiol. 27:477-492 (1998)).Both pflB and tdcE from E. coli require the presence of pyruvate formatelyase activating enzyme, encoded by pflA. Further, a short proteinencoded by yfiD in E. coli can associate with and restore activity tooxygen-cleaved pyruvate formate lyase (Vey et al., Proc. Natl. Acad.Sci. U.S.A. 105:16137-16141 (2008). Note that pflA and pflB from E. coliwere expressed in S. cerevisiae as a means to increase cytosolicacetyl-CoA for butanol production as described in WO/2008/080124].Additional pyruvate formate lyase and activating enzyme candidates,encoded by pfl and act, respectively, are found in Clostridiumpasteurianum (Weidner et al., J. Bacteriol. 178:2440-2444 (1996)).

Further, different enzymes can be used in combination to convertpyruvate into acetyl-CoA. For example, in S. cerevisiae, acetyl-CoA isobtained in the cytosol by first decarboxylating pyruvate to formacetaldehyde; the latter is oxidized to acetate by acetaldehydedehydrogenase and subsequently activated to form acetyl-CoA byacetyl-CoA synthetase. Acetyl-CoA synthetase is a native enzyme inseveral other organisms including E. coli (Kumari et al., J. Bacteriol.177:2878-2886 (1995)), Salmonella enterica (Starai et al., Microbiology151:3793-3801 (2005); Starai et al., J. Biol. Chem. 280:26200-26205(2005)), and Moorella thermoacetica (described already). Alternatively,acetate can be activated to form acetyl-CoA by acetate kinase andphosphotransacetylase. Acetate kinase first converts acetate intoacetyl-phosphate with the accompanying use of an ATP molecule.Acetyl-phosphate and CoA are next converted into acetyl-CoA with therelease of one phosphate by phosphotransacetylase. Both acetate kinaseand phosphotransacetylyase are well-studied enzymes in severalClostridia and Methanosarcina thermophile.

Yet another way of converting pyruvate to acetyl-CoA is via pyruvateoxidase. Pyruvate oxidase converts pyruvate into acetate, usingubiquione as the electron acceptor. In E. coli, this activity is encodedby poxB. PoxB has similarity to pyruvate decarboxylase of S. cerevisiaeand Zymomonas mobilis. The enzyme has a thiamin pyrophosphate cofactor(Koland and Gennis, Biochemistry 21:4438-4442 (1982)); O'Brien et al.,Biochemistry 16:3105-3109 (1977); O'Brien and Gennis, J. Biol. Chem.255:3302-3307 (1980)) and a flavin adenine dinucleotide (FAD) cofactor.Acetate can then be converted into acetyl-CoA by either acetyl-CoAsynthetase or by acetate kinase and phosphotransacetylase, as describedearlier. Some of these enzymes can also catalyze the reverse reactionfrom acetyl-CoA to pyruvate.

For enzymes that use reducing equivalents in the form of NADH or NADPH,these reduced carriers can be generated by transferring electrons fromreduced ferredoxin. Two enzymes catalyze the reversible transfer ofelectrons from reduced ferredoxins to NAD(P)⁺, ferredoxin:NAD⁺oxidoreductase (EC 1.18.1.3) and ferredoxin:NADP⁺ oxidoreductase (FNR,EC 1.18.1.2). Ferredoxin:NADP⁺ oxidoreductase (FNR, EC 1.18.1.2) has anoncovalently bound FAD cofactor that facilitates the reversibletransfer of electrons from NADPH to low-potential acceptors such asferredoxins or flavodoxins (Blaschkowski et al., Eur. J. Biochem.123:563-569 (1982); Fujii et al., 1977). The Helicobacter pylori FNR,encoded by HP1164 (fqrB), is coupled to the activity ofpyruvate:ferredoxin oxidoreductase (PFOR) resulting in thepyruvate-dependent production of NADPH (St et al. 2007). An analogousenzyme is found in Campylobacter jejuni (St et al. 2007). Aferredoxin:NADP⁺ oxidoreductase enzyme is encoded in the E. coli genomeby fpr (Bianchi et al. 1993). Ferredoxin:NAD⁺ oxidoreductase utilizesreduced ferredoxin to generate NADH from NAD⁺. In several organisms,including E. coli, this enzyme is a component of multifunctionaldioxygenase enzyme complexes. The ferredoxin:NAD⁺ oxidoreductase of E.coli, encoded by hcaD, is a component of the 3-phenylproppionatedioxygenase system involved in involved in aromatic acid utilization(Diaz et al. 1998). NADH:ferredoxin reductase activity was detected incell extracts of Hydrogenobacter thermophilus strain TK-6, although agene with this activity has not yet been indicated (Yoon et al. 2006).Finally, the energy-conserving membrane-associated Rnf-type proteins(Seedorf et al., Proc. Natl. Acad. Sci. U.S.A. 105:2128-2133 (2008);Herrmann et al., J. Bacteriol. 190:784-791 (2008)) provide a means togenerate NADH or NADPH from reduced ferredoxin. Additionalferredoxin:NAD(P)+ oxidoreductases have been annotated in Clostridiumcarboxydivorans P7.

Protein GenBank ID GI Number Organism HP1164 NP_207955.1 15645778Helicobacter pylori RPA3954 CAE29395.1 39650872 Rhodopseudomonaspalustris fpr BAH29712.1 225320633 Hydrogenobacter thermophilus yumCNP_391091.2 255767736 Bacillus subtilis CJE0663 AAW35824.1 57167045Campylobacter jejuni fpr P28861.4 399486 Escherichia coli hcaDAAC75595.1 1788892 Escherichia coli LOC100282643 NP_001149023.1226497434 Zea mays RnfC EDK33306.1 146346770 Clostridium kluyveri RnfDEDK33307.1 146346771 Clostridium kluyveri RnfG EDK33308.1 146346772Clostridium kluyveri RnfE EDK33309.1 146346773 Clostridium kluyveri RnfAEDK33310.1 146346774 Clostridium kluyveri RnfB EDK33311.1 146346775Clostridium kluyveri CcarbDRAFT_2639 ZP_05392639.1 255525707 Clostridiumcarboxidivorans P7 CcarbDRAFT_2638 ZP_05392638.1 255525706 Clostridiumcarboxidivorans P7 CcarbDRAFT_2636 ZP_05392636.1 255525704 Clostridiumcarboxidivorans P7 CcarbDRAFT_5060 ZP_05395060.1 255528241 Clostridiumcarboxidivorans P7 CcarbDRAFT_2450 ZP_05392450.1 255525514 Clostridiumcarboxidivorans P7 CcarbDRAFT_1084 ZP_05391084.1 255524124 Clostridiumcarboxidivorans P7

Ferredoxins are small acidic proteins containing one or more iron-sulfurclusters that function as intracellular electron carriers with a lowreduction potential. Reduced ferredoxins donate electrons toFe-dependent enzymes such as ferredoxin-NADP⁺ oxidoreductase,pyruvate:ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxinoxidoreductase (OFOR). The H. thermophilus gene fdx1 encodes a[4Fe-4S]-type ferredoxin that is required for the reversiblecarboxylation of 2-oxoglutarate and pyruvate by OFOR and PFOR,respectively (Yamamoto et al., Extremophiles 14:79-85 (2010)). Theferredoxin associated with the Sulfolobus solfataricus2-oxoacid:ferredoxin reductase is a monomeric dicluster [3Fe-4S][4Fe-4S]type ferredoxin (Park et al. 2006). While the gene associated with thisprotein has not been fully sequenced, the N-terminal domain shares 93%homology with the zfx ferredoxin from S. acidocaldarius. The E. coligenome encodes a soluble ferredoxin of unknown physiological function,fdx. Some evidence indicates that this protein can function iniron-sulfur cluster assembly (Takahashi and Nakamura, 1999). Additionalferredoxin proteins have been characterized in Helicobacter pylori(Mukhopadhyay et al. 2003) and Campylobacter jejuni (van Vliet et al.2001). A 2Fe-2S ferredoxin from Clostridium pasteurianum has been clonedand expressed in E. coli (Fujinaga and Meyer, Biochemical andBiophysical Research Communications, 192(3): (1993)). Acetogenicbacteria such as Moorella thermoacetica, Clostridium carboxidivorans P7and Rhodospirillum rubrum are predicted to encode several ferredoxins,listed below.

Protein GenBank ID GI Number Organism fdx1 BAE02673.1 68163284Hydrogenobacter thermophilus M11214.1 AAA83524.1 144806 Clostridiumpasteurianum Zfx AAY79867.1 68566938 Sulfolobus acidocalarius FdxAAC75578.1 1788874 Escherichia coli hp_0277 AAD07340.1 2313367Helicobacter pylori fdxA CAL34484.1 112359698 Campylobacter jejuniMoth_0061 ABC18400.1 83571848 Moorella thermoacetica Moth_1200ABC19514.1 83572962 Moorella thermoacetica Moth_1888 ABC20188.1 83573636Moorella thermoacetica Moth_2112 ABC20404.1 83573852 Moorellathermoacetica Moth_1037 ABC19351.1 83572799 Moorella thermoaceticaCcarbDRAFT_4383 ZP_05394383.1 255527515 Clostridium carboxidivorans P7CcarbDRAFT_2958 ZP_05392958.1 255526034 Clostridium carboxidivorans P7CcarbDRAFT_2281 ZP_05392281.1 255525342 Clostridium carboxidivorans P7CcarbDRAFT_5296 ZP_05395295.1 255528511 Clostridium carboxidivorans P7CcarbDRAFT_1615 ZP_05391615.1 255524662 Clostridium carboxidivorans P7CcarbDRAFT_1304 ZP_05391304.1 255524347 Clostridium carboxidivorans P7cooF AAG29808.1 11095245 Carboxydothermus hydrogenoformans fdxNCAA35699.1 46143 Rhodobacter capsulatus Rru_A2264 ABC23064.1 83576513Rhodospirillum rubrum Rru_A1916 ABC22716.1 83576165 Rhodospirillumrubrum Rru_A2026 ABC22826.1 83576275 Rhodospirillum rubrum cooFAAC45122.1 1498747 Rhodospirillum rubrum fdxN AAA26460.1 152605Rhodospirillum rubrum Alvin_2884 ADC63789.1 288897953 Allochromatiumvinosum DSM 180 fdx YP_002801146.1 226946073 Azotobacter vinelandii DJCKL_3790 YP_001397146.1 153956381 Clostridium kluyveri DSM 555 fer1NP_949965.1 39937689 Rhodopseudomonas palustris CGA009 fdx CAA12251.13724172 Thauera aromatica CHY_2405 YP_361202.1 78044690 Carboxydothermushydrogenoformans fer YP_359966.1 78045103 Carboxydothermushydrogenoformans fer AAC83945.1 1146198 Bacillus subtilis fdx1NP_249053.1 15595559 Pseudomonas aeruginosa PA01 yfhL AP_003148.189109368 Escherichia coli K-12

Succinyl-CoA transferase catalyzes the conversion of succinyl-CoA tosuccinate while transferring the CoA moiety to a CoA acceptor molecule.Many transferases have broad specificity and can utilize CoA acceptorsas diverse as acetate, succinate, propionate, butyrate,2-methylacetoacetate, 3-ketohexanoate, 3-ketopentanoate, valerate,crotonate, 3-mercaptopropionate, propionate, vinylacetate, and butyrate,among others.

The conversion of succinate to succinyl-CoA can be carried by atransferase which does not require the direct consumption of an ATP orGTP. This type of reaction is common in a number of organisms. Theconversion of succinate to succinyl-CoA can also be catalyzed bysuccinyl-CoA:Acetyl-CoA transferase. The gene product of cat1 ofClostridium kluyveri has been shown to exhibit succinyl-CoA:acetyl-CoAtransferase activity (Sohling and Gottschalk, J. Bacteriol. 178:871-880(1996)). In addition, the activity is present in Trichomonas vaginalis(van Grinsven et al. 2008) and Trypanosoma brucei (Riviere et al. 2004).The succinyl-CoA:acetate CoA-transferase from Acetobacter aceti, encodedby aarC, replaces succinyl-CoA synthetase in a variant TCA cycle(Mullins et al. 2008). Similar succinyl-CoA transferase activities arealso present in Trichomonas vaginalis (van Grinsven et al. 2008),Trypanosoma brucei (Riviere et al. 2004) and Clostridium kluyveri(Sohling and Gottschalk, 1996c). The beta-ketoadipate:succinyl-CoAtransferase encoded by pcaI and pcaJ in Pseudomonas putida is yetanother candidate (Kaschabek et al. 2002). The aforementioned proteinsare identified below.

Protein GenBank ID GI Number Organism cat1 P38946.1 729048 Clostridiumkluyveri TVAG_395550 XP_001330176 123975034 Trichomonas vaginalis G3Tb11.02.0290 XP_828352 71754875 Trypanosoma brucei pcaI AAN69545.124985644 Pseudomonas putida pcaJ NP_746082.1 26990657 Pseudomonas putidaaarC ACD85596.1 189233555 Acetobacter aceti

An additional exemplary transferase that converts succinate tosuccinyl-CoA while converting a 3-ketoacyl-CoA to a 3-ketoacid issuccinyl-CoA:3:ketoacid-CoA transferase (EC 2.8.3.5). Exemplarysuccinyl-CoA:3:ketoacid-CoA transferases are present in Helicobacterpylori (Corthesy-Theulaz et al. 1997), Bacillus subtilis, and Homosapiens (Fukao et al. 2000; Tanaka et al. 2002). The aforementionedproteins are identified below.

Protein GenBank ID GI Number Organism HPAG1_0676 YP_627417 108563101Helicobacter pylori HPAG1_0677 YP_627418 108563102 Helicobacter pyloriScoA NP_391778 16080950 Bacillus subtilis ScoB NP_391777 16080949Bacillus subtilis OXCT1 NP_000427 4557817 Homo sapiens OXCT2 NP_07140311545841 Homo sapiens

Converting succinate to succinyl-CoA by succinyl-CoA:3:ketoacid-CoAtransferase requires the simultaneous conversion of a 3-ketoacyl-CoAsuch as acetoacetyl-CoA to a 3-ketoacid such as acetoacetate. Conversionof a 3-ketoacid back to a 3-ketoacyl-CoA can be catalyzed by anacetoacetyl-CoA:acetate:CoA transferase. Acetoacetyl-CoA:acetate:CoAtransferase converts acetoacetyl-CoA and acetate to acetoacetate andacetyl-CoA, or vice versa. Exemplary enzymes include the gene productsof atoAD from E. coli (Hanai et al., Appl Environ Microbiol 73:7814-7818(2007), ctfAB from C. acetobutylicum (Jojima et al., Appl MicrobiolBiotechnol 77:1219-1224 (2008), and ctfAB from Clostridiumsaccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem.71:58-68 (2007)) are shown below.

Protein GenBank ID GI Number Organism AtoA NP_416726.1 2492994Escherichia coli AtoD NP_416725.1 2492990 Escherichia coli CtfANP_149326.1 15004866 Clostridium acetobutylicum CtfB NP_149327.115004867 Clostridium acetobutylicum CtfA AAP42564.1 31075384 Clostridiumsaccharoperbutylacetonicum CtfB AAP42565.1 31075385 Clostridiumsaccharoperbutylacetonicum

Yet another possible CoA acceptor is benzylsuccinate.Succinyl-CoA:(R)-Benzylsuccinate CoA-Transferase functions as part of ananaerobic degradation pathway for toluene in organisms such as Thaueraaromatics (Leutwein and Heider, J. Bact. 183(14) 4288-4295 (2001)).Homologs can be found in Azoarcus sp. T, Aromatoleum aromaticum EbN1,and Geobacter metallireducens GS-15. The aforementioned proteins areidentified below.

Protein GenBank ID GI Number Organism bbsE AAF89840 9622535 Thaueraaromatic Bbsf AAF89841 9622536 Thauera aromatic bbsE AAU45405.1 52421824Azoarcus sp. T bbsF AAU45406.1 52421825 Azoarcus sp. T bbsE YP_158075.156476486 Aromatoleum aromaticum EbN1 bbsF YP_158074.1 56476485Aromatoleum aromaticum EbN1 Gmet_1521 YP_384480.1 78222733 Geobactermetallireducens GS-15 Gmet_1522 YP_384481.1 78222734 Geobactermetallireducens GS-15

Additionally, yell encodes a propionyl CoA:succinate CoA transferase inE. coli (Haller et al., Biochemistry, 39(16) 4622-4629). Close homologscan be found in, for example, Citrobacter youngae ATCC 29220, Salmonellaenterica subsp. arizonae serovar, and Yersinia intermedia ATCC 29909.The aforementioned proteins are identified below.

Protein GenBank ID GI Number Organism ygfH NP_417395.1 16130821Escherichia coli str. K-12 substr. MG1655 CIT292_04485 ZP_03838384.1227334728 Citrobacter youngae ATCC 29220 SARI_04582 YP_001573497.1161506385 Salmonella enterica subsp. arizonae serovar yinte0001_14430ZP_04635364.1 238791727 Yersinia intermedia ATCC 29909

Citrate lyase (EC 4.1.3.6) catalyzes a series of reactions resulting inthe cleavage of citrate to acetate and oxaloacetate. The enzyme isactive under anaerobic conditions and is composed of three subunits: anacyl-carrier protein (ACP, gamma), an ACP transferase (alpha), and aacyl lyase (beta). Enzyme activation uses covalent binding andacetylation of an unusual prosthetic group,2′-(5″-phosphoribosyl)-3-′-dephospho-CoA, which is similar in structureto acetyl-CoA. Acylation is catalyzed by CitC, a citrate lyasesynthetase. Two additional proteins, CitG and CitX, are used to convertthe apo enzyme into the active holo enzyme (Schneider et al.,Biochemistry 39:9438-9450 (2000)). Wild type E. coli does not havecitrate lyase activity; however, mutants deficient in molybdenumcofactor synthesis have an active citrate lyase (Clark, FEMS Microbiol.Lett. 55:245-249 (1990)). The E. coli enzyme is encoded by citEFD andthe citrate lyase synthetase is encoded by citC (Nilekani and SivaRaman,Biochemistry 22:4657-4663 (1983)). The Leuconostoc mesenteroides citratelyase has been cloned, characterized and expressed in E. coli (Bekal etal., J. Bacteriol. 180:647-654 (1998)). Citrate lyase enzymes have alsobeen identified in enterobacteria that utilize citrate as a carbon andenergy source, including Salmonella typhimurium and Klebsiellapneumoniae (Bott, Arch. Microbiol. 167: 78-88 (1997); Bott and Dimroth,Mol. Microbiol. 14:347-356 (1994)). The aforementioned proteins aretabulated below.

Protein GenBank ID GI Number Organism citF AAC73716.1 1786832Escherichia coli Cite AAC73717.2 87081764 Escherichia coli citDAAC73718.1 1786834 Escherichia coli citC AAC73719.2 87081765 Escherichiacoli citG AAC73714.1 1786830 Escherichia coli citX AAC73715.1 1786831Escherichia coli citF CAA71633.1 2842397 Leuconostoc mesenteroides CiteCAA71632.1 2842396 Leuconostoc mesenteroides citD CAA71635.1 2842395Leuconostoc mesenteroides citC CAA71636.1 3413797 Leuconostocmesenteroides citG CAA71634.1 2842398 Leuconostoc mesenteroides citXCAA71634.1 2842398 Leuconostoc mesenteroides citF NP_459613.1 16763998Salmonella typhimurium cite AAL19573.1 16419133 Salmonella typhimuriumcitD NP_459064.1 16763449 Salmonella typhimurium citC NP_459616.116764001 Salmonella typhimurium citG NP_459611.1 16763996 Salmonellatyphimurium citX NP_459612.1 16763997 Salmonella typhimurium citFCAA56217.1 565619 Klebsiella pneumoniae cite CAA56216.1 565618Klebsiella pneumoniae citD CAA56215.1 565617 Klebsiella pneumoniae citCBAH66541.1 238774045 Klebsiella pneumoniae citG CAA56218.1 565620Klebsiella pneumoniae citX AAL60463.1 18140907 Klebsiella pneumoniae

Acetate kinase (EC 2.7.2.1) catalyzes the reversible ATP-dependentphosphorylation of acetate to acetylphosphate. Exemplary acetate kinaseenzymes have been characterized in many organisms including E. coli,Clostridium acetobutylicum and Methanosarcina thermophile (Ingram-Smithet al., J. Bacteriol. 187:2386-2394 (2005); Fox and Roseman, J. Biol.Chem. 261:13487-13497 (1986); Winzer et al., Microbioloy 143 (Pt10):3279-3286 (1997)). Acetate kinase activity has also beendemonstrated in the gene product of E. coli purT (Marolewski et al.,Biochemistry 33:2531-2537 (1994). Some butyrate kinase enzymes (EC2.7.2.7), for example buk1 and buk2 from Clostridium acetobutylicum,also accept acetate as a substrate (Hartmanis, M. G., J. Biol. Chem.262:617-621 (1987)).

Protein GenBank ID GI Number Organism ackA NP_416799.1 16130231Escherichia coli Ack AAB18301.1 1491790 Clostridium acetobutylicum AckAAA72042.1 349834 Methanosarcina thermophila purT AAC74919.1 1788155Escherichia coli buk1 NP_349675 15896326 Clostridium acetobutylicum buk2Q97II1 20137415 Clostridium acetobutylicum

The formation of acetyl-CoA from acetylphosphate is catalyzed byphosphotransacetylase (EC 2.3.1.8). The pta gene from E. coli encodes anenzyme that reversibly converts acetyl-CoA into acetyl-phosphate(Suzuki, T., Biochim. Biophys. Acta 191:559-569 (969)). Additionalacetyltransferase enzymes have been characterized in Bacillus subtilis(Rado and Hoch, Biochim. Biophys. Acta 321:114-125 (1973), Clostridiumkluyveri (Stadtman, E., Methods Enzymol. 1:5896-599 (1955), andThermotoga maritima (Bock et al., J. Bacteriol. 181:1861-1867 (1999)).This reaction is also catalyzed by some phosphotranbutyrylase enzymes(EC 2.3.1.19) including the ptb gene products from Clostridiumacetobutylicum (Wiesenborn et al., App. Environ. Microbiol. 55:317-322(1989); Walter et al., Gene 134:107-111 (1993)). Additional ptb genesare found in butyrate-producing bacterium L2-50 (Louis et al., J.Bacteriol. 186:2099-2106 (2004) and Bacillus megaterium (Vazquez et al.,Curr. Microbiol. 42:345-349 (2001).

Protein GenBank ID GI Number Organism Pta NP_l 416800.1 71152910Escherichia coli Pta P39646 730415 Bacillus subtilis Pta A5N801146346896 Clostridium kluyveri Pta Q9X0L4 6685776 Thermotoga maritimaPtb NP_349676 34540484 Clostridium acetobutylicum Ptb AAR19757.138425288 butyrate-producing bacterium L2-50 Ptb CAC07932.1 10046659Bacillus megaterium

The acylation of acetate to acetyl-CoA is catalyzed by enzymes withacetyl-CoA synthetase activity. Two enzymes that catalyze this reactionare AMP-forming acetyl-CoA synthetase (EC 6.2.1.1) and ADP-formingacetyl-CoA synthetase (EC 6.2.1.13). AMP-forming acetyl-CoA synthetase(ACS) is the predominant enzyme for activation of acetate to acetyl-CoA.Exemplary ACS enzymes are found in E. coli (Brown et al., J. Gen.Microbiol. 102:327-336 (1977)), Ralstonia eutropha (Priefert andSteinbuchel, J. Bacteriol. 174:6590-6599 (1992)), Methanothermobacterthermautotrophicus (Ingram-Smith and Smith, Archaea 2:95-107 (2007)),Salmonella enterica (Gulick et al., Biochemistry 42:2866-2873 (2003))and Saccharomyces cerevisiae (Jogl and Tong, Biochemistry 43:1425-1431(2004)). ADP-forming acetyl-CoA synthetases are reversible enzymes witha generally broad substrate range (Musfeldt and Schonheit, J. Bacteriol.184:636-644 (2002)). Two isozymes of ADP-forming acetyl-CoA synthetasesare encoded in the Archaeoglobus fulgidus genome by are encoded byAF1211 and AF1983 (Musfeldt and Schonheit, supra (2002)). The enzymefrom Haloarcula marismortui (annotated as a succinyl-CoA synthetase)also accepts acetate as a substrate and reversibility of the enzyme wasdemonstrated (Brasen and Schonheit, Arch. Microbiol. 182:277-287(2004)). The ACD encoded by PAE3250 from hyperthermophilic crenarchaeonPyrobaculum aerophilum showed the broadest substrate range of allcharacterized ACDs, reacting with acetate, isobutyryl-CoA (preferredsubstrate) and phenylacetyl-CoA (Brasen and Schonheit, supra (2004)).Directed evolution or engineering can be used to modify this enzyme tooperate at the physiological temperature of the host organism. Theenzymes from A. fulgidus, H. marismortui and P. aerophilum have all beencloned, functionally expressed, and characterized in E. coli (Brasen andSchonheit, supra (2004); Musfeldt and Schonheit, supra (2002)).Additional candidates include the succinyl-CoA synthetase encoded bysucCD in E. coli (Buck et al., Biochemistry 24:6245-6252 (1985)) and theacyl-CoA ligase from Pseudomonas putida (Fernandez-Valverde et al.,Appl. Environ. Microbiol. 59:1149-1154 (1993)). The aforementionedproteins are tabulated below.

Protein GenBank ID GI Number Organism acs AAC77039.1 1790505 Escherichiacoli acoE AAA21945.1 141890 Ralstonia eutropha acs1 ABC87079.1 86169671Methanothermobacter thermautotrophicus acs1 AAL23099.1 16422835Salmonella enterica ACS1 Q01574.2 257050994 Saccharomyces cerevisiaeAF1211 NP_070039.1 11498810 Archaeoglobus fulgidus AF1983 NP_070807.111499565 Archaeoglobus fulgidus scs YP_135572.1 55377722 Haloarculamarismortui PAE3250 NP_560604.1 18313937 Pyrobaculum aerophilum str. IM2sucC NP_415256.1 16128703 Escherichia coli sucD AAC73823.1 1786949Escherichia coli paaF AAC24333.2 22711873 Pseudomonas putida

The product yields per C-mol of substrate of microbial cellssynthesizing reduced fermentation products such as methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate,are limited by insufficient reducing equivalents in the carbohydratefeedstock. Reducing equivalents, or electrons, can be extracted fromsynthesis gas components such as CO and H₂ using carbon monoxidedehydrogenase (CODH) and hydrogenase enzymes, respectively. The reducingequivalents are then passed to acceptors such as oxidized ferredoxins,oxidized quinones, oxidized cytochromes, NAD(P)+, water, or hydrogenperoxide to form reduced ferredoxin, reduced quinones, reducedcytochromes, NAD(P)H, H₂, or water, respectively. Reduced ferredoxin andNAD(P)H are particularly useful as they can serve as redox carriers forvarious Wood-Ljungdahl pathway and reductive TCA cycle enzymes.

Additional redox availability from CO and/or H₂ can improve the yieldsof reduced products such as methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate. The maximumtheoretical yield to produce methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate from glucose is 1.33mole MAA per mole of glucose under aerobic conditions via the pathwaysdisclosed herein:C₆H₁₂O₆→1.33C₄H₆O₂+0.67CO₂+2H₂O

When both feedstocks of sugar and syngas are available, the syngascomponents CO and H₂ can be utilized to generate reducing equivalents byemploying the hydrogenase and CO dehydrogenase. The reducing equivalentsgenerated from syngas components will be utilized to power the glucoseto methacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate production pathways. Theoretically, all carbons inglucose will be conserved, thus resulting in a maximal theoretical yieldto produce methacrylic acid, methacrylate ester, 3-hydroxyisobutyrateand/or 2-hydroxyisobutyrate from glucose at 2 mol methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate permol of glucose under either aerobic or anaerobic conditions as describedherein.1C₆H₁₂O₆+2CO₂+6H₂→2C₄H₆O₂+6H₂OOr1C₆H₁₂O₆+2CO+4H₂→2C₄H₆O₂+4H₂O

As shown in above example, a combined feedstock strategy where syngas iscombined with a sugar-based feedstock or other carbon substrate cangreatly improve the theoretical yields. In this co-feeding approach,syngas components H₂ and CO can be utilized by the hydrogenase and COdehydrogenase to generate reducing equivalents, that can be used topower chemical production pathways in which the carbons from sugar orother carbon substrates will be maximally conserved and the theoreticalyields improved. In case of methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate production from glucoseor sugar, the theoretical yields improve from 1.33 mol methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate permol of glucose to 2 mol methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate per mol of glucose.Such improvements provide environmental and economic benefits andgreatly enhance sustainable chemical production.

Herein the enzymes and the corresponding genes used for extracting redoxfrom syngas components are described. CODH is a reversible enzyme thatinterconverts CO and CO₂ at the expense or gain of electrons. Thenatural physiological role of the CODH in ACS/CODH complexes is toconvert CO₂ to CO for incorporation into acetyl-CoA by acetyl-CoAsynthase. Nevertheless, such CODH enzymes are suitable for theextraction of reducing equivalents from CO due to the reversible natureof such enzymes. Expressing such CODH enzymes in the absence of ACSallows them to operate in the direction opposite to their naturalphysiological role (i.e., CO oxidation).

In M. thermoacetica, C. hydrogenoformans, C. carboxidivorans P7, andseveral other organisms, additional CODH encoding genes are locatedoutside of the ACS/CODH operons. These enzymes provide a means forextracting electrons (or reducing equivalents) from the conversion ofcarbon monoxide to carbon dioxide. The M. thermoacetica gene (GenbankAccession Number: YP_(—)430813) is expressed by itself in an operon andis believed to transfer electrons from CO to an external mediator likeferredoxin in a “Ping-pong” reaction. The reduced mediator then couplesto other reduced nicolinamide adenine dinucleotide phosphate (NAD(P)H)carriers or ferredoxin-dependent cellular processes (Ragsdale, Annals ofthe New York Academy of Sciences 1125: 129-136 (2008)). The genesencoding the C. hydrogenoformans CODH-II and CooF, a neighboringprotein, were cloned and sequenced (Gonzalez and Robb, FEMS MicrobiolLett. 191:243-247 (2000)). The resulting complex was membrane-bound,although cytoplasmic fractions of CODH-II were shown to catalyze theformation of NADPH suggesting an anabolic role (Svetlitchnyi et al., JBacteriol. 183:5134-5144 (2001)). The crystal structure of the CODH-IIis also available (Dobbek et al., Science 293:1281-1285 (2001)). SimilarACS-free CODH enzymes can be found in a diverse array of organismsincluding Geobacter metallireducens GS-15, Chlorobium phaeobacteroidesDSM 266, Clostridium cellulolyticum H10, Desulfovibrio desulfuricanssubsp. desulfuricans str. ATCC 27774, Pelobacter carbinolicus DSM 2380,and Campylobacter curvus 525.92.

Protein GenBank ID GI Number Organism CODH (putative) YP_430813 83590804Moorella thermoacetica CODH-II (CooS-II) YP_358957 78044574Carboxydothermus hydrogenoformans CooF YP_358958 78045112Carboxydothermus hydrogenoformans CODH (putative) ZP_05390164.1255523193 Clostridium carboxidivorans P7 CcarbDRAFT_0341 ZP_05390341.1255523371 Clostridium carboxidivorans P7 CcarbDRAFT_1756 ZP_05391756.1255524806 Clostridium carboxidivorans P7 CcarbDRAFT_2944 ZP_05392944.1255526020 Clostridium carboxidivorans P7 CODH YP_384856.1 78223109Geobacter metallireducens GS-15 Cpha266_0148 YP_910642.1 119355998Chlorobium phaeobacteroides (cytochrome c) DSM 266 Cpha266_0149 (CODH)YP_910643.1 119355999 Chlorobium phaeobacteroides DSM 266 Ccel_0438YP_002504800.1 220927891 Clostridium cellulolyticum H10 Ddes_0382 (CODH)YP_002478973.1 220903661 Desulfovibrio desulfuricans subsp.desulfuricans str. ATCC 27774 Ddes 0381 (CooC) YP_002478972.1 220903660Desulfovibrio desulfuricans subsp. desulfuricans str. ATCC 27774Pcar_0057 (CODH) YP_355490.1 7791767 Pelobacter carbinolicus DSM 2380Pcar_0058 (CooC) YP_355491.1 7791766 Pelobacter carbinolicus DSM 2380Pcar_0058 (HypA) YP_355492.1 7791765 Pelobacter carbinolicus DSM 2380CooS (CODH) YP_001407343.1 154175407 Campylobacter curvus 525.92

In some cases, hydrogenase encoding genes are located adjacent to aCODH. In Rhodospirillum rubrum, the encoded CODH/hydrogenase proteinsform a membrane-bound enzyme complex that has been indicated to be asite where energy, in the form of a proton gradient, is generated fromthe conversion of CO and H₂O to CO₂ and H₂ (Fox et al., J. Bacteriol.178:6200-6208 (1996)). The CODH-I of C. hydrogenoformans and itsadjacent genes have been proposed to catalyze a similar functional rolebased on their similarity to the R. rubrum CODH/hydrogenase gene cluster(Wu et al., PLoS Genet. 1:e65 (2005)). The C. hydrogenoformans CODH-Iwas also shown to exhibit intense CO oxidation and CO₂ reductionactivities when linked to an electrode (Parkin et al., J Am. Chem. Soc.129:10328-10329 (2007)). The protein sequences of exemplary CODH andhydrogenase genes can be identified by the following GenBank accessionnumbers.

Protein GenBank ID GI Number Organism CODH-I YP_360644 78043418Carboxydothermus (CooS-I) hydrogenoformans CooF YP_360645 78044791Carboxydothermus hydrogenoformans HypA YP_360646 78044340Carboxydothermus hydrogenoformans CooH YP_360647 78043871Carboxydothermus hydrogenoformans CooU YP_360648 78044023Carboxydothermus hydrogenoformans CooX YP_360649 78043124Carboxydothermus hydrogenoformans CooL YP_360650 78043938Carboxydothermus hydrogenoformans CooK YP_360651 78044700Carboxydothermus hydrogenoformans CooM YP_360652 78043942Carboxydothermus hydrogenoformans CooC YP_360654.1 78043296Carboxydothermus hydrogenoformans CooA-1 YP_360655.1 78044021Carboxydothermus hydrogenoformans CooL AAC45118 1515468 Rhodospirillumrubrum CooX AAC45119 1515469 Rhodospirillum rubrum CooU AAC45120 1515470Rhodospirillum rubrum CooH AAC45121 1498746 Rhodospirillum rubrum CooFAAC45122 1498747 Rhodospirillum rubrum CODH AAC45123 1498748Rhodospirillum rubrum (CooS) CooC AAC45124 1498749 Rhodospirillum rubrumCooT AAC45125 1498750 Rhodospirillum rubrum CooJ AAC45126 1498751Rhodospirillum rubrum

Native to E. coli and other enteric bacteria are multiple genes encodingup to four hydrogenases (Sawers, G., Antonie Van Leeuwenhoek 66:57-88(1994); Sawers et al., J. Bacteriol. 164:1324-1331 (1985); Sawers andBoxer, Eur. J Biochem. 156:265-275 (1986); Sawers et al., J Bacteriol.168:398-404 (1986)). Given the multiplicity of enzyme activities, E.coli or another host organism can provide sufficient hydrogenaseactivity to split incoming molecular hydrogen and reduce thecorresponding acceptor. E. coli possesses two uptake hydrogenases, Hyd-1and Hyd-2, encoded by the hyaABCDEF and hybOABCDEFG gene clusters,respectively (Lukey et al., How E. coli is equipped to oxidize hydrogenunder different redox conditions, J Biol Chem published online Nov. 16,2009). Hyd-1 is oxygen-tolerant, irreversible, and is coupled to quinonereduction via the hyaC cytochrome. Hyd-2 is sensitive to O₂, reversible,and transfers electrons to the periplasmic ferredoxin hybA which, inturn, reduces a quinone via the hybB integral membrane protein. Reducedquinones can serve as the source of electrons for fumarate reductase inthe reductive branch of the TCA cycle. Reduced ferredoxins can be usedby enzymes such as NAD(P)H:ferredoxin oxidoreductases to generate NADPHor NADH. They can alternatively be used as the electron donor forreactions such as pyruvate ferredoxin oxidoreductase, AKG ferredoxinoxidoreductase, and 5,10-methylene-H4folate reductase.

Protein GenBank ID GI Number Organism HyaA AAC74057.1 1787206Escherichia coli HyaB AAC74058.1 1787207 Escherichia coli HyaCAAC74059.1 1787208 Escherichia coli HyaD AAC74060.1 1787209 Escherichiacoli HyaE AAC74061.1 1787210 Escherichia coli HyaF AAC74062.1 1787211Escherichia coli

Protein GenBank ID GI Number Organism HybO AAC76033.1 1789371Escherichia coli HybA AAC76032.1 1789370 Escherichia coli HybBAAC76031.1 2367183 Escherichia coli HybC AAC76030.1 1789368 Escherichiacoli HybD AAC76029.1 1789367 Escherichia coli HybE AAC76028.1 1789366Escherichia coli HybF AAC76027.1 1789365 Escherichia coli HybGAAC76026.1 1789364 Escherichia coli

The hydrogen-lyase systems of E. coli include hydrogenase 3, amembrane-bound enzyme complex using ferredoxin as an acceptor, andhydrogenase 4 that also uses a ferredoxin acceptor. Hydrogenase 3 and 4are encoded by the hyc and hyf gene clusters, respectively. Hydrogenase3 has been shown to be a reversible enzyme (Maeda et al., Appl MicrobiolBiotechnol 76(5):1035-42 (2007)). Hydrogenase activity in E. coli isalso dependent upon the expression of the hyp genes whose correspondingproteins are involved in the assembly of the hydrogenase complexes(Jacobi et al., Arch. Microbiol 158:444-451 (1992); Rangarajan et al.,J. Bacteriol, 190:1447-1458 (2008)).

Protein GenBank ID GI Number Organism HycA NP_417205 16130632Escherichia coli HycB NP_417204 16130631 Escherichia coli HycC NP_41720316130630 Escherichia coli HycD NP_417202 16130629 Escherichia coli HycENP_417201 16130628 Escherichia coli HycF NP_417200 16130627 Escherichiacoli HycG NP_417199 16130626 Escherichia coli HycH NP_417198 16130625Escherichia coli HycI NP_417197 16130624 Escherichia coli

Protein GenBank ID GI Number Organism HyfA NP_416976 90111444Escherichia coli HyfB NP_416977 16130407 Escherichia coli HyfC NP_41697890111445 Escherichia coli HyfD NP_416979 16130409 Escherichia coli HyfENP_416980 16130410 Escherichia coli HyfF NP_416981 16130411 Escherichiacoli HyfG NP_416982 16130412 Escherichia coli HyfH NP_416983 16130413Escherichia coli HyfI NP_416984 16130414 Escherichia coli HyfJ NP_41698590111446 Escherichia coli HyfR NP_416986 90111447 Escherichia coli

Protein GenBank ID GI Number Organism HypA NP_417206 16130633Escherichia coli HypB NP_417207 16130634 Escherichia coli HypC NP_41720816130635 Escherichia coli HypD NP_417209 16130636 Escherichia coli HypENP_417210 226524740 Escherichia coli HypF NP_417192 16130619 Escherichiacoli

The M. thermoacetica hydrogenases are suitable for a host that lackssufficient endogenous hydrogenase activity. M. thermoacetica can growwith CO₂ as the exclusive carbon source indicating that reducingequivalents are extracted from H₂ to enable acetyl-CoA synthesis via theWood-Ljungdahl pathway (Drake, H. L., J. Bacteriol. 150:702-709 (1982);Drake and Daniel, Res. Microbiol. 155:869-883 (2004); Kellum and Drake,J. Bacteriol. 160:466-469 (1984)) (see FIGS. 12 and 13). M.thermoacetica has homologs to several hyp, hyc, and hyf genes from E.coli. The protein sequences encoded for by these genes are identified bythe following GenBank accession numbers.

Proteins in M. thermoacetica whose genes are homologous to the E. colihyp genes are shown below.

Protein GenBank ID GI Number Organism Moth_2175 YP_431007 83590998Moorella thermoacetica Moth_2176 YP_431008 83590999 Moorellathermoacetica Moth_2177 YP_431009 83591000 Moorella thermoaceticaMoth_2178 YP_431010 83591001 Moorella thermoacetica Moth_2179 YP_43101183591002 Moorella thermoacetica Moth_2180 YP_431012 83591003 Moorellathermoacetica Moth_2181 YP_431013 83591004 Moorella thermoacetica

Proteins in M. thermoacetica that are homologous to the E. coliHydrogenase 3 and/or 4 proteins are listed below.

Protein GenBank ID GI Number Organism Moth_2182 YP_431014 83591005Moorella thermoacetica Moth_2183 YP_431015 83591006 Moorellathermoacetica Moth_2184 YP_431016 83591007 Moorella thermoaceticaMoth_2185 YP_431017 83591008 Moorella thermoacetica Moth_2186 YP_43101883591009 Moorella thermoacetica Moth_2187 YP_431019 83591010 Moorellathermoacetica Moth_2188 YP_431020 83591011 Moorella thermoaceticaMoth_2189 YP_431021 83591012 Moorella thermoacetica Moth_2190 YP_43102283591013 Moorella thermoacetica Moth_2191 YP_431023 83591014 Moorellathermoacetica Moth_2192 YP_431024 83591015 Moorella thermoacetica

In addition, several gene clusters encoding hydrogenase functionalityare present in M. thermoacetica and their corresponding proteinsequences are provided below.

Protein GenBank ID GI Number Organism Moth_0439 YP_429313 83589304Moorella thermoacetica Moth_0440 YP_429314 83589305 Moorellathermoacetica Moth_0441 YP_429315 83589306 Moorella thermoaceticaMoth_0442 YP_429316 83589307 Moorella thermoacetica Moth_0809 YP_42967083589661 Moorella thermoacetica Moth_0810 YP_429671 83589662 Moorellathermoacetica Moth_0811 YP_429672 83589663 Moorella thermoaceticaMoth_0812 YP_429673 83589664 Moorella thermoacetica Moth_0814 YP_42967483589665 Moorella thermoacetica Moth_0815 YP_429675 83589666 Moorellathermoacetica Moth_0816 YP_429676 83589667 Moorella thermoaceticaMoth_1193 YP_430050 83590041 Moorella thermoacetica Moth_1194 YP_43005183590042 Moorella thermoacetica Moth_1195 YP_430052 83590043 Moorellathermoacetica Moth_1196 YP_430053 83590044 Moorella thermoaceticaMoth_1717 YP_430562 83590553 Moorella thermoacetica Moth_1718 YP_43056383590554 Moorella thermoacetica Moth_1719 YP_430564 83590555 Moorellathermoacetica Moth_1883 YP_430726 83590717 Moorella thermoaceticaMoth_1884 YP_430727 83590718 Moorella thermoacetica Moth_1885 YP_43072883590719 Moorella thermoacetica Moth_1886 YP_430729 83590720 Moorellathermoacetica Moth_1887 YP_430730 83590721 Moorella thermoaceticaMoth_1888 YP_430731 83590722 Moorella thermoacetica Moth_1452 YP_43030583590296 Moorella thermoacetica Moth_1453 YP_430306 83590297 Moorellathermoacetica Moth_1454 YP_430307 83590298 Moorella thermoacetica

Ralstonia eutropha H16 uses hydrogen as an energy source with oxygen asa terminal electron acceptor. Its membrane-bound uptake[NiFe]-hydrogenase is an “O2-tolerant” hydrogenase (Cracknell, et al.Proc Nat Acad Sci, 106(49) 20681-20686 (2009)) that isperiplasmically-oriented and connected to the respiratory chain via ab-type cytochrome (Schink and Schlegel, Biochim. Biophys. Acta, 567,315-324 (1979); Bernhard et al., Eur. J. Biochem. 248, 179-186 (1997)).R. eutropha also contains an O₂-tolerant soluble hydrogenase encoded bythe Hox operon which is cytoplasmic and directly reduces NAD+ at theexpense of hydrogen (Schneider and Schlegel, Biochim. Biophys. Acta 452,66-80 (1976); Burgdorf, J. Bact. 187(9) 3122-3132 (2005)). Solublehydrogenase enzymes are additionally present in several other organismsincluding Geobacter sulfurreducens (Coppi, Microbiology 151, 1239-1254(2005)), Synechocystis str. PCC 6803 (Germer, J. Biol. Chem., 284(52),36462-36472 (2009)), and Thiocapsa roseopersicina (Rakhely, Appl.Environ. Microbiol. 70(2) 722-728 (2004)). The Synechocystis enzyme iscapable of generating NADPH from hydrogen. Overexpression of both theHox operon from Synechocystis str. PCC 6803 and the accessory genesencoded by the Hyp operon from Nostoc sp. PCC 7120 led to increasedhydrogenase activity compared to expression of the Hox genes alone(Germer, J. Biol. Chem. 284(52), 36462-36472 (2009)).

Protein GenBank ID GI Number Organism HoxF NP_942727.1 38637753Ralstonia eutropha H16 HoxU NP_942728.1 38637754 Ralstonia eutropha H16HoxY NP_942729.1 38637755 Ralstonia eutropha H16 HoxH NP_942730.138637756 Ralstonia eutropha H16 HoxW NP_942731.1 38637757 Ralstoniaeutropha H16 HoxI NP_942732.1 38637758 Ralstonia eutropha H16 HoxENP_953767.1 39997816 Geobacter sulfurreducens HoxF NP_953766.1 39997815Geobacter sulfurreducens HoxU NP_953765.1 39997814 Geobactersulfurreducens HoxY NP_953764.1 39997813 Geobacter sulfurreducens HoxHNP_953763.1 39997812 Geobacter sulfurreducens GSU2717 NP_953762.139997811 Geobacter sulfurreducens HoxE NP_441418.1 16330690Synechocystis str. PCC 6803 HoxF NP_441417.1 16330689 Synechocystis str.PCC 6803 Unknown NP_441416.1 16330688 Synechocystis str. PCC function6803 HoxU NP_441415.1 16330687 Synechocystis str. PCC 6803 HoxYNP_441414.1 16330686 Synechocystis str. PCC 6803 Unknown NP_441413.116330685 Synechocystis str. PCC function 6803 Unknown NP_441412.116330684 Synechocystis str. PCC function 6803 HoxH NP_441411.1 16330683Synechocystis str. PCC 6803 HypF NP_484737.1 17228189 Nostoc sp. PCC7120 HypC NP_484738.1 17228190 Nostoc sp. PCC 7120 HypD NP_484739.117228191 Nostoc sp. PCC 7120 Unknown NP_484740.1 17228192 Nostoc sp. PCC7120 function HypE NP_484741.1 17228193 Nostoc sp. PCC 7120 HypANP_484742.1 17228194 Nostoc sp. PCC 7120 HypB NP_484743.1 17228195Nostoc sp. PCC 7120 Hox1E AAP50519.1 37787351 Thiocapsa roseopersicinaHox1F AAP50520.1 37787352 Thiocapsa roseopersicina Hox1U AAP50521.137787353 Thiocapsa roseopersicina Hox1Y AAP50522.1 37787354 Thiocapsaroseopersicina Hox1H AAP50523.1 37787355 Thiocapsa roseopersicina

Several enzymes and the corresponding genes used for fixing carbondioxide to either pyruvate or phosphoenolpyruvate to form the TCA cycleintermediates, oxaloacetate or malate are described below.

Carboxylation of phosphoenolpyruvate to oxaloacetate is catalyzed byphosphoenolpyruvate carboxylase. Exemplary PEP carboxylase enzymes areencoded by ppc in E. coli (Kai et al., Arch. Biochem. Biophys.414:170-179 (2003), ppcA in Methylobacterium extorquens AM1 (Arps etal., J. Bacteriol. 175:3776-3783 (1993), and ppc in Corynebacteriumglutamicum (Eikmanns et al., Mol. Gen. Genet. 218:330-339 (1989).

Protein GenBank ID GI Number Organism Ppc NP_418391 16131794 Escherichiacoli ppcA AAB58883 28572162 Methylobacterium extorquens Ppc ABB5327080973080 Corynebacterium glutamicum

An alternative enzyme for converting phosphoenolpyruvate to oxaloacetateis PEP carboxykinase, which simultaneously forms an ATP whilecarboxylating PEP. In most organisms PEP carboxykinase serves agluconeogenic function and converts oxaloacetate to PEP at the expenseof one ATP. S. cerevisiae is one such organism whose native PEPcarboxykinase, PCKJ, serves a gluconeogenic role (Valdes-Hevia et al.,FEBS Lett. 258:313-316 (1989). E. coli is another such organism, as therole of PEP carboxykinase in producing oxaloacetate is believed to beminor when compared to PEP carboxylase, which does not form ATP,possibly due to the higher K_(m) for bicarbonate of PEP carboxykinase(Kim et al., Appl. Environ. Microbiol. 70:1238-1241 (2004)).Nevertheless, activity of the native E. coli PEP carboxykinase from PEPtowards oxaloacetate has been recently demonstrated in ppc mutants of E.coli K-12 (Kwon et al., J. Microbiol. Biotechnol. 16:1448-1452 (2006)).These strains exhibited no growth defects and had increased succinateproduction at high NaHCO₃ concentrations. Mutant strains of E. coli canadopt Pck as the dominant CO2-fixing enzyme following adaptive evolution(Zhang et al. 2009). In some organisms, particularly rumen bacteria, PEPcarboxykinase is quite efficient in producing oxaloacetate from PEP andgenerating ATP. Examples of PEP carboxykinase genes that have beencloned into E. coli include those from Mannheimia succiniciproducens(Lee et al., Biotechnol. Bioprocess Eng. 7:95-99 (2002)),Anaerobiospirillum succiniciproducens (Laivenieks et al., Appl. Environ.Microbiol. 63:2273-2280 (1997), and Actinobacillus succinogenes (Kim etal. supra). The PEP carboxykinase enzyme encoded by Haemophilusinfluenza is effective at forming oxaloacetate from PEP.

Protein GenBank ID GI Number Organism PCK1 NP_013023 6322950Saccharomyces cerevisiae pck NP_417862.1 16131280 Escherichia coli pckAYP_089485.1 52426348 Mannheimia succiniciproducens pckA O09460.1 3122621Anaerobiospirillum succiniciproducens pckA Q6W6X5 75440571Actinobacillus succinogenes pckA P43923.1 1172573 Haemophilus influenza

Pyruvate carboxylase (EC 6.4.1.1) directly converts pyruvate tooxaloacetate at the cost of one ATP. Pyruvate carboxylase enzymes areencoded by PYC1 (Walker et al., Biochem. Biophys. Res. Commun.176:1210-1217 (1991) and PYC2 (Walker et al., supra) in Saccharomycescerevisiae, and pyc in Mycobacterium smegmatis (Mukhopadhyay andPurwantini, Biochim. Biophys. Acta 1475:191-206 (2000)).

Protein GenBank ID GI Number Organism PYC1 NP_011453 6321376Saccharomyces cerevisiae PYC2 NP_009777 6319695 Saccharomyces cerevisiaePyc YP_890857.1 118470447 Mycobacterium smegmatis

Malic enzyme can be applied to convert CO₂ and pyruvate to malate at theexpense of one reducing equivalent. Malic enzymes for this purpose caninclude, without limitation, malic enzyme (NAD-dependent) and malicenzyme (NADP-dependent). For example, one of the E. coli malic enzymes(Takeo, J. Biochem. 66:379-387 (1969)) or a similar enzyme with higheractivity can be expressed to enable the conversion of pyruvate and CO₂to malate. By fixing carbon to pyruvate as opposed to PEP, malic enzymeallows the high-energy phosphate bond from PEP to be conserved bypyruvate kinase whereby ATP is generated in the formation of pyruvate orby the phosphotransferase system for glucose transport. Although malicenzyme is typically assumed to operate in the direction of pyruvateformation from malate, overexpression of the NAD-dependent enzyme,encoded by maeA, has been demonstrated to increase succinate productionin E. coli while restoring the lethal Δpfl-ΔldhA phenotype underanaerobic conditions by operating in the carbon-fixing direction (Stolsand Donnelly, Appl. Environ. Microbiol. 63(7) 2695-2701 (1997)). Asimilar observation was made upon overexpressing the malic enzyme fromAscaris suum in E. coli (Stols et al., Appl. Biochem. Biotechnol.63-65(1), 153-158 (1997)). The second E. coli malic enzyme, encoded bymaeB, is NADP-dependent and also decarboxylates oxaloacetate and otheralpha-keto acids (Iwakura et al., J. Biochem. 85(5):1355-65 (1979)).

Protein GenBank ID GI Number Organism maeA NP_415996 90111281Escherichia coli maeB NP_416958 16130388 Escherichia coli NAD-ME P27443126732 Ascaris suum

The enzymes used for converting oxaloacetate (formed from, for example,PEP carboxylase, PEP carboxykinase, or pyruvate carboxylase) or malate(formed from, for example, malic enzyme or malate dehydrogenase) tosuccinyl-CoA via the reductive branch of the TCA cycle are malatedehydrogenase, fumarate dehydratase (fumarase), fumarate reductase, andsuccinyl-CoA transferase. The genes for each of the enzymes aredescribed herein above.

Enzymes, genes and methods for engineering pathways from succinyl-CoA tovarious products into a microorganism are now known in the art. Theadditional reducing equivalents obtained from CO and/or H₂, as disclosedherein, improve the yields of methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate when utilizingcarbohydrate-based feedstock. For example, methacrylic acid,methacrylate ester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate canbe produced from succinyl-CoA (see Figures). Exemplary enzymes for theconversion succinyl-CoA to methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate include enzymesdisclosed herein.

Enzymes, genes and methods for engineering pathways from glycolysisintermediates to various products into a microorganism are known in theart. The additional reducing equivalents obtained from CO and H₂, asdescribed herein, improve the yields of all these products oncarbohydrates. For example, methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate can be produced fromthe glycolysis intermediate, succinate. Exemplary enzymes for theconversion of succinate to MAA include succinyl-CoA transferase,succinyl-CoA synthetase, succinyl-CoA reductase (aldehyde forming),4-hydroxybutyrate dehydrogenase, 4-hydroxybutyrate kinase,phosphotrans-4-hydroxybutyrylase, succinate reductase, succinyl-CoAreductase (alcohol forming), 4-hydroxybutyryl-CoA synthetase,4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA mutase,3-hydroxyisobutyryl-CoA synthetase, 3-hydroxyisobutyryl-CoA transferase,3-hydroxyisobutyryl-CoA hydrolase, 3-hydroxyisobutyrate dehydratase,3-hydroxyisobutyryl-CoA dehydratase, methacrylyl-CoA synthetase,methacrylyl-CoA transferase and methacrylyl-CoA hydrolase.

EXAMPLE XVI Methods for Handling CO and Anaerobic Cultures

This example describes methods used in handling CO and anaerobiccultures.

A. Handling of CO in Small Quantities for Assays and Small Cultures.

CO is an odorless, colorless and tasteless gas that is a poison.Therefore, cultures and assays that utilized CO required specialhandling. Several assays, including CO oxidation, acetyl-CoA synthesis,CO concentration using myoglobin, and CO tolerance/utilization in smallbatch cultures, called for small quantities of the CO gas that weredispensed and handled within a fume hood. Biochemical assays called forsaturating very small quantities (<2 mL) of the biochemical assay mediumor buffer with CO and then performing the assay. All of the CO handlingsteps were performed in a fume hood with the sash set at the properheight and blower turned on; CO was dispensed from a compressed gascylinder and the regulator connected to a Schlenk line. The latterensures that equal concentrations of CO were dispensed to each ofseveral possible cuvettes or vials. The Schlenk line was set upcontaining an oxygen scrubber on the input side and an oil pressurerelease bubbler and vent on the other side. Assay cuvettes were bothanaerobic and CO-containing, Therefore, the assay cuvettes were tightlysealed with a rubber stopper and reagents were added or removed usinggas-tight needles and syringes. Secondly, small (˜50 mL) cultures weregrown with saturating CO in tightly stoppered serum bottles. As with thebiochemical assays, the CO-saturated microbial cultures wereequilibrated in the fume hood using the Schlenk line setup. Both thebiochemical assays and microbial cultures were in portable, sealedcontainers and in small volumes making for safe handling outside of thefume hood. The compressed CO tank was adjacent to the fume hood.

Typically, a Schlenk line was used to dispense CO to cuvettes, eachvented. Rubber stoppers on the cuvettes were pierced with 19 or 20 gagedisposable syringe needles and were vented with the same. An oil bubblerwas used with a CO tank and oxygen scrubber. The glass or quartzspectrophotometer cuvettes have a circular hole on top into which aKontes stopper sleeve, Sz7 774250-0007 was fitted. The CO detector unitwas positioned proximal to the fume hood.

B. Handling of CO in Larger Quantities Fed to Large-Scale Cultures.

Fermentation cultures are fed either CO or a mixture of CO and H₂ tosimulate syngas as a feedstock in fermentative production. Therefore,quantities of cells ranging from 1 liter to several liters can includethe addition of CO gas to increase the dissolved concentration of CO inthe medium. In these circumstances, fairly large and continuouslyadministered quantities of CO gas are added to the cultures. Atdifferent points, the cultures are harvested or samples removed.Alternatively, cells are harvested with an integrated continuous flowcentrifuge that is part of the fermenter.

The fermentative processes are carried out under anaerobic conditions.In some cases, it is uneconomical to pump oxygen or air into fermentersto ensure adequate oxygen saturation to provide a respiratoryenvironment. In addition, the reducing power generated during anaerobicfermentation may be needed in product formation rather than respiration.Furthermore, many of the enzymes for various pathways areoxygen-sensitive to varying degrees. Classic acetogens such as M.thermoacetica are obligate anaerobes and the enzymes in theWood-Ljungdahl pathway are highly sensitive to irreversible inactivationby molecular oxygen. While there are oxygen-tolerant acetogens, therepertoire of enzymes in the Wood-Ljungdahl pathway might beincompatible in the presence of oxygen because most are metallo-enzymes,key components are ferredoxins, and regulation can divert metabolismaway from the Wood-Ljungdahl pathway to maximize energy acquisition. Atthe same time, cells in culture act as oxygen scavengers that moderatethe need for extreme measures in the presence of large cell growth.

C. Anaerobic Chamber and Conditions.

Exemplary anaerobic chambers are available commercially (see, forexample, Vacuum Atmospheres Company, Hawthorne Calif.; MBraun,Newburyport Mass.). Conditions included an O₂ concentration of 1 ppm orless and 1 atm pure N₂. In one example, 3 oxygen scrubbers/catalystregenerators were used, and the chamber included an O₂ electrode (suchas Teledyne; City of Industry Calif.). Nearly all items and reagentswere cycled four times in the airlock of the chamber prior to openingthe inner chamber door. Reagents with a volume >5 mL were sparged withpure N₂ prior to introduction into the chamber. Gloves are changedtwice/yr and the catalyst containers were regenerated periodically whenthe chamber displays increasingly sluggish response to changes in oxygenlevels. The chamber's pressure was controlled through one-way valvesactivated by solenoids. This feature allowed setting the chamberpressure at a level higher than the surroundings to allow transfer ofvery small tubes through the purge valve.

The anaerobic chambers achieved levels of O₂ that were consistently verylow and were needed for highly oxygen sensitive anaerobic conditions.However, growth and handling of cells does not usually require suchprecautions. In an alternative anaerobic chamber configuration, platinumor palladium can be used as a catalyst that requires some hydrogen gasin the mix. Instead of using solenoid valves, pressure release can becontrolled by a bubbler. Instead of using instrument-based O₂monitoring, test strips can be used instead.

D. Anaerobic Microbiology.

Small cultures were handled as described above for CO handling. Inparticular, serum or media bottles are fitted with thick rubber stoppersand aluminum crimps are employed to seal the bottle. Medium, such asTerrific Broth, is made in a conventional manner and dispensed to anappropriately sized serum bottle. The bottles are sparged with nitrogenfor ˜30 min of moderate bubbling. This removes most of the oxygen fromthe medium and, after this step, each bottle is capped with a rubberstopper (such as Bellco 20 mm septum stoppers; Bellco, Vineland, N.J.)and crimp-sealed (Bellco 20 mm). Then the bottles of medium areautoclaved using a slow (liquid) exhaust cycle. At least sometimes aneedle can be poked through the stopper to provide exhaust duringautoclaving; the needle needs to be removed immediately upon removalfrom the autoclave. The sterile medium has the remaining mediumcomponents, for example buffer or antibiotics, added via syringe andneedle. Prior to addition of reducing agents, the bottles areequilibrated for 30-60 minutes with nitrogen (or CO depending upon use).A reducing agent such as a 100×150 mM sodium sulfide, 200 mMcysteine-HCl is added. This is made by weighing the sodium sulfide intoa dry beaker and the cysteine into a serum bottle, bringing both intothe anaerobic chamber, dissolving the sodium sulfide into anaerobicwater, then adding this to the cysteine in the serum bottle. The bottleis stoppered immediately as the sodium sulfide solution generateshydrogen sulfide gas upon contact with the cysteine. When injecting intothe culture, a syringe filter is used to sterilize the solution. Othercomponents are added through syringe needles, such as B12 (10 μMcyanocobalamin), nickel chloride (NiCl₂, 20 microM final concentrationfrom a 40 mM stock made in anaerobic water in the chamber and sterilizedby autoclaving or by using a syringe filter upon injection into theculture), and ferrous ammonium sulfate (final concentration needed is100 μM—made as 100-1000× stock solution in anaerobic water in thechamber and sterilized by autoclaving or by using a syringe filter uponinjection into the culture). To facilitate faster growth under anaerobicconditions, the 1 liter bottles were inoculated with 50 mL of apreculture grown anaerobically. Induction of the pA1-lacO1 promoter inthe vectors was performed by addition of isopropylβ-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mMand was carried out for about 3 hrs.

Large cultures can be grown in larger bottles using continuous gasaddition while bubbling. A rubber stopper with a metal bubbler is placedin the bottle after medium addition and sparged with nitrogen for 30minutes or more prior to setting up the rest of the bottle. Each bottleis put together such that a sterile filter will sterilize the gasbubbled in and the hoses on the bottles are compressible with small Cclamps. Medium and cells are stirred with magnetic stir bars. Once allmedium components and cells are added, the bottles are incubated in anincubator in room air but with continuous nitrogen sparging into thebottles.

EXAMPLE XVIII CO Oxidation (CODH) Assay

This example describes assay methods for measuring CO oxidation (COdehydrogenase; CODH).

The 7 gene CODH/ACS operon of Moorella thermoacetica was cloned into E.coli expression vectors. The intact ˜10 kbp DNA fragment was cloned, andit is likely that some of the genes in this region are expressed fromtheir own endogenous promoters and all contain endogenous ribosomalbinding sites. These clones were assayed for CO oxidation, using anassay that quantitatively measures CODH activity. Antisera to the M.thermoacetica gene products was used for Western blots to estimatespecific activity. M. thermoacetica is Gram positive, and ribosomebinding site elements are expected to work well in E. coli. Thisactivity, described below in more detail, was estimated to be ˜ 1/50thof the M. thermoacetica specific activity. It is possible that CODHactivity of recombinant E. coli cells could be limited by the fact thatM. thermoacetica enzymes have temperature optima around 55° C.Therefore, a mesophilic CODH/ACS pathway could be advantageous such asthe close relative of Moorella that is mesophilic and does have anapparently intact CODH/ACS operon and a Wood-Ljungdahl pathway,Desulfitobacterium hafniense. Acetogens as potential host organismsinclude, but are not limited to, Rhodospirillum rubrum, Moorellathermoacetica and Desulfitobacterium hafniense.

CO oxidation is both the most sensitive and most robust of the CODH/ACSassays. It is likely that an E. coli-based syngas using system willultimately need to be about as anaerobic as Clostridial (i.e., Moorella)systems, especially for maximal activity. Improvement in CODH should bepossible but will ultimately be limited by the solubility of CO gas inwater.

Initially, each of the genes was cloned individually into expressionvectors. Combined expression units for multiple subunits/1 complex weregenerated. Expression in E. coli at the protein level was determined.Both combined M. thermoacetica CODH/ACS operons and individualexpression clones were made.

CO oxidation assay. This assay is one of the simpler, reliable, and moreversatile assays of enzymatic activities within the Wood-Ljungdahlpathway and tests CODH (Seravalli et al., Biochemistry 43:3944-3955(2004)). A typical activity of M. thermoacetica CODH specific activityis 500 U at 55° C. or ˜60 U at 25° C. This assay employs reduction ofmethyl viologen in the presence of CO. This is measured at 578 nm instoppered, anaerobic, glass cuvettes.

In more detail, glass rubber stoppered cuvettes were prepared afterfirst washing the cuvette four times in deionized water and one timewith acetone. A small amount of vacuum grease was smeared on the top ofthe rubber gasket. The cuvette was gassed with CO, dried 10 min with a22 Ga. needle plus an exhaust needle. A volume of 0.98 mL of reactionbuffer (50 mM Hepes, pH 8.5, 2 mM dithiothreitol (DTT) was added using a22 Ga. needle, with exhaust needled, and 100% CO. Methyl viologen (CH₃viologen) stock was 1 M in water. Each assay used 20 microliters for 20mM final concentration. When methyl viologen was added, an 18 Ga needle(partial) was used as a jacket to facilitate use of a Hamilton syringeto withdraw the CH₃ viologen. 4-5 aliquots were drawn up and discardedto wash and gas equilibrate the syringe. A small amount of sodiumdithionite (0.1 M stock) was added when making up the CH₃ viologen stockto slightly reduce the CH₃ viologen. The temperature was equilibrated to55° C. in a heated Olis spectrophotometer (Bogart GA). A blank reaction(CH₃ viologen+buffer) was run first to measure the base rate of CH₃viologen reduction. Crude E. coli cell extracts of ACS90 and ACS91(CODH-ACS operon of M. thermoacetica with and without, respectively, thefirst cooC). 10 microliters of extract were added at a time, mixed andassayed. Reduced CH₃ viologen turns purple. The results of an assay areshown in Table 2.

TABLE 2 Crude extract CO Oxidation Activities. ACS90 7.7 mg/ml ACS9111.8 mg/ml Mta98 9.8 mg/ml Mta99 11.2 mg/ml Extract Vol OD/ U/ml U/mgACS90 10 microliters 0.073 0.376 0.049 ACS91 10 microliters 0.096 0.4940.042 Mta99 10 microliters 0.0031 0.016 0.0014 ACS90 10 microliters0.099 0.51 0.066 Mta99 25 microliters 0.012 0.025 0.0022 ACS91 25microliters 0.215 0.443 0.037 Mta98 25 microliters 0.019 0.039 0.004ACS91 10 microliters 0.129 0.66 0.056 Averages ACS90 0.057 U/mg ACS910.045 U/mg Mta99 0.0018 U/mg

Mta98/Mta99 are E. coli MG1655 strains that express methanolmethyltransferase genes from M. thermoacetia and, therefore, arenegative controls for the ACS90 ACS91 E. coli strains that contain M.thermoacetica CODH operons.

If ˜1% of the cellular protein is CODH, then these figures would beapproximately 100× less than the 500 U/mg activity of pure M.thermoacetica CODH. Actual estimates based on Western blots are 0.5% ofthe cellular protein, so the activity is about 50× less than for M.thermoacetica CODH. Nevertheless, this experiment demonstrates COoxidation activity in recombinant E. coli with a much smaller amount inthe negative controls. The small amount of CO oxidation (CH₃ viologenreduction) seen in the negative controls indicates that E. coli may havea limited ability to reduce CH₃ viologen.

To estimate the final concentrations of CODH and Mtr proteins, SDS-PAGEfollowed by Western blot analyses were performed on the same cellextracts used in the CO oxidation, ACS, methyltransferase, and corrinoidFe—S assays. The antisera used were polyclonal to purified M.thermoacetica CODH-ACS and Mtr proteins and were visualized using analkaline phosphatase-linked goat-anti-rabbit secondary antibody. TheWesterns were performed and results are shown in FIG. 14. The amounts ofCODH in ACS90 and ACS91 were estimated at 50 ng by comparison to thecontrol lanes. Expression of CODH-ACS operon genes including 2 CODHsubunits and the methyltransferase were confirmed via Western blotanalysis. Therefore, the recombinant E. coli cells express multiplecomponents of a 7 gene operon. In addition, both the methyltransferaseand corrinoid iron sulfur protein were active in the same recombinant E.coli cells. These proteins are part of the same operon cloned into thesame cells.

The CO oxidation assays were repeated using extracts of Moorellathermoacetica cells for the positive controls. Though CODH activity inE. coli ACS90 and ACS91 was measurable, it was at about 130-150× lowerthan the M. thermoacetica control. The results of the assay are shown inFIG. 15. Briefly, cells (M. thermoacetica or E. coli with the CODH/ACSoperon; ACS90 or ACS91 or empty vector: pZA33S) were grown and extractsprepared as described above. Assays were performed as described above at55° C. at various times on the day the extracts were prepared. Reductionof methylviologen was followed at 578 nm over a 120 sec time course.

These results describe the CO oxidation (CODH) assay and results.Recombinant E. coli cells expressed CO oxidation activity as measured bythe methyl viologen reduction assay.

EXAMPLE XVIII E. coli CO Tolerance Experiment and CO Concentration Assay(Myoglobin Assay)

This example describes the tolerance of E. coli for high concentrationsof CO.

To test whether or not E. coli can grow anaerobically in the presence ofsaturating amounts of CO, cultures were set up in 120 ml serum bottleswith 50 ml of Terrific Broth medium (plus reducing solution, NiCl₂,Fe(II)NH₄SO₄, cyanocobalamin, IPTG, and chloramphenicol) as describedabove for anaerobic microbiology in small volumes. One half of thesebottles were equilibrated with nitrogen gas for 30 min. and one half wasequilibrated with CO gas for 30 min. An empty vector (pZA33) was used asa control, and cultures containing the pZA33 empty vector as well asboth ACS90 and ACS91 were tested with both N₂ and CO. All wereinoculated and grown for 36 hrs with shaking (250 rpm) at 37° C. At theend of the 36 hour period, examination of the flasks showed high amountsof growth in all. The bulk of the observed growth occurred overnightwith a long lag.

Given that all cultures appeared to grow well in the presence of CO, thefinal CO concentrations were confirmed. This was performed using anassay of the spectral shift of myoglobin upon exposure to CO. Myoglobinreduced with sodium dithionite has an absorbance peak at 435 nm; thispeak is shifted to 423 nm with CO. Due to the low wavelength and need torecord a whole spectrum from 300 nm on upwards, quartz cuvettes must beused. CO concentration is measured against a standard curve and dependsupon the Henry's Law constant for CO of maximum water solubility=970micromolar at 20° C. and 1 atm.

For the myoglobin test of CO concentration, cuvettes were washed 10×with water, 1× with acetone, and then stoppered as with the CODH assay.N₂ was blown into the cuvettes for ˜10 min. A volume of 1 ml ofanaerobic buffer (HEPES, pH 8.0, 2 mM DTT) was added to the blank (notequilibrated with CO) with a Hamilton syringe. A volume of 10 microlitermyoglobin (˜1 mM—can be varied, just need a fairly large amount) and 1microliter dithionite (20 mM stock) were added. A CO standard curve wasmade using CO saturated buffer added at 1 microliter increments. Peakheight and shift was recorded for each increment. The cultures testedwere pZA33/CO, ACS90/CO, and ACS91/CO. Each of these was added in 1microliter increments to the same cuvette. Midway through the experimenta second cuvette was set up and used. The results are shown in Table 3.

TABLE 3 Carbon Monoxide Concentrations, 36 hrs. Strain and Growth FinalCO concentration Conditions (micromolar) pZA33-CO 930 ACS90-CO 638 494734 883 ave 687 SD 164 ACS91-CO 728 812 760 611 ave. 728 SD 85

The results shown in Table 3 indicate that the cultures grew whether ornot a strain was cultured in the presence of CO or not. These resultsindicate that E. coli can tolerate exposure to CO under anaerobicconditions and that E. coli cells expressing the CODH-ACS operon canmetabolize some of the CO.

These results demonstrate that E. coli cells, whether expressingCODH/ACS or not, were able to grow in the presence of saturating amountsof CO. Furthermore, these grew equally well as the controls in nitrogenin place of CO. This experiment demonstrated that laboratory strains ofE. coli are insensitive to CO at the levels achievable in a syngasproject performed at normal atmospheric pressure. In addition,preliminary experiments indicated that the recombinant E. coli cellsexpressing CODH/ACS actually consumed some CO, probably by oxidation tocarbon dioxide.

EXAMPLE XVIX Pathways for the Production of MAA and 3-HydroxyisobutyricAcid from Succinate Via Methylmalonyl-CoA

The reductive TCA cycle improves the yield of methacrylic acid (MAA)when utilizing a carbohydrate-based feedstock. MAA production can beachieved in a recombinant organism by the pathway shown in FIG. 16A.

For example, MAA and/or 3-hydroxyisobutyric acid can be produced fromsuccinate via a methylmalonyl-CoA intermediate as shown in FIG. 16A.Exemplary enzymes for the conversion of succinate to MAA or3-hydroxyisobutyric acid by this route include succinyl-CoA transferase,succinyl-CoA synthetase, methylmalonyl-CoA mutase, methylmalonyl-CoAepimerase, methylmalonyl-CoA reductase, methylmalonate semialdehydereductase and 3-hydroxyisobutyrate dehydratase.

In this pathway, central metabolic intermediates are first channeledinto succinate. For formation of succinate, phosphoenolpyruvate (PEP) isconverted into oxaloacetate either via PEP carboxykinase or PEPcarboxylase. Alternatively, PEP is converted first to pyruvate bypyruvate kinase and then to oxaloacetate by methylmalonyl-CoAcarboxytransferase or pyruvate carboxylase. Oxaloacetate is thenconverted to succinate by means of the reductive TCA cycle.

Succinate is then activated to succinyl-CoA by a succinyl-CoAtransferase or synthetase. Methylmalonyl-CoA mutase then formsmethylmalonyl-CoA from succinyl-CoA. Methylmalonyl-CoA is then reducedto methylmalonate semialdehyde. Further reduction of methylmalonatesemialdehyde yields 3-hydroxyisobutyric acid, which can be secreted as aproduct or further transformed to MAA via dehydration.

Exemplary enzyme candidates for the transformations shown in FIG. 16Aare described below. Succinyl-CoA transferase and synthetase enzymeswere described previously in Example XV (RTCA cycle).

Methylmalonyl-CoA Mutase.

Methylmalonyl-CoA mutase is a cobalamin-dependent enzyme that convertssuccinyl-CoA to methylmalonyl-CoA. In E. coli, the reversibleadenosylcobalamin-dependant mutase participates in a three-step pathwayleading to the conversion of succinate to propionate. Exemplary MCMenzymes are described in Example V.

Alternatively, isobutyryl-CoA mutase (ICM) could catalyze the proposedtransformation. ICM is a cobalamin-dependent methylmutase in the MCMfamily that reversibly rearranges the carbon backbone of butyryl-CoAinto isobutyryl-CoA (Ratnatilleke, J Biol. Chem. 274:31679-31685(1999)). Exemplary ICM enzymes are described in Example VII.

Methylmalonyl-CoA Epimerase.

Methylmalonyl-CoA epimerase (MMCE) is the enzyme that interconverts(R)-methylmalonyl-CoA and (S)-methylmalonyl-CoA. MMCE is an essentialenzyme in the breakdown of odd-numbered fatty acids and of the aminoacids valine, isoleucine, and methionine. Exemplary MMCE enzymes aredescribed in Example V.

Methylmalonyl-CoA Reductase.

The reduction of methylmalonyl-CoA to its corresponding aldehyde,methylmalonate semialdehyde, is catalyzed by a CoA-dependent aldehydedehydrogenase. Exemplary enzymes are described in Example V.

Methylmalonate Semialdehyde Reductase.

The reduction of methylmalonate semialdehyde to 3-hydroxyisobutyrate iscatalyzed by methylmalonate semialdehyde reductase or3-hydroxyisobutyrate dehydrogenase. This enzyme participates in valine,leucine and isoleucine degradation and has been identified in bacteria,eukaryotes, and mammals. Exemplary methylmalonate semialdehyde reductaseenzymes are described in Example V.

3-Hydroxyisobutyrate Dehydratase.

The dehydration of 3-hydroxyisobutyrate to methylacrylic acid iscatalyzed by an enzyme with 3-hydroxyisobutyrate dehydratase activity.Exemplary 3-hydroxyisobutyrate dehydratase enzymes are described inExample V.

An active reductive TCA cycle improves the yield of the acetyl-CoAderived product methacrylic acid (MAA), as shown in the exemplarypathways of FIG. 17. Pathways of the reductive TCA cycle are describedherein as well as the conversion of succinyl-CoA to methacrylic acid(see also FIGS. 3 and 16A and Example V). FIGS. 17A and 17B showexemplary pathways. The enzymatic transformations are carried out by theenzymes as shown. FIG. 17A shows the pathways for fixation of CO₂ tosuccinyl-CoA using the reductive TCA cycle. FIG. 17B shows exemplarypathways for the biosynthesis of 3-hydroxyisobutric acid and methacrylicacid from succinyl-CoA; the enzymatic transformations shown are carriedout by the following enzymes: A. Methylmalonyl-CoA mutase, B.Methylmalonyl-CoA epimerase, C. Methylmalonyl-CoA reductase, D.Methylmalonate semialdehyde reductase, E. 3-Hydroxyisobutyratedehydratase.

EXAMPLE XX Pathways for the Production of MAA and 3-HydroxyisobutyricAcid from Succinate Via 4-Hydroxybutyryl-CoA

The reductive TCA cycle improves the yield of methacrylic acid (MAA)when utilizing a carbohydrate-based feedstock. MAA production can beachieved in a recombinant organism by as shown in FIG. 16B.

For example, MAA and/or 3-hydroxyisobutyric acid can be produced fromsuccinate via a 4-hydroxybutyryl-CoA intermediate by a number ofalternate routes depicted in FIG. 16B. Exemplary enzymes for theseroutes include: succinyl-CoA transferase, succinyl-CoA synthetase,succinyl-CoA reductase (aldehyde forming), 4-hydroxybutyratedehydrogenase, 4-hydroxybutyrate kinase,phosphotrans-4-hydroxybutyrylase, succinate reductase, succinyl-CoAreductase (alcohol forming), 4-hydroxybutyryl-CoA synthetase,4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA mutase,3-hydroxyisobutyryl-CoA synthetase, 3-hydroxyisobutyryl-CoA transferase,3-hydroxyisobutyryl-CoA hydrolase, 3-hydroxyisobutyrate dehydratase,3-hydroxyisobutyryl-CoA dehydratase, methacrylyl-CoA synthetase,methacrylyl-CoA transferase and methacrylyl-CoA hydrolase.

Formation of the 4-hydroxybutyryl-CoA intermediate from succinateproceeds through several routes depicted in FIG. 16B. Succinate is firstconverted to succinyl-CoA by a CoA transferase or synthetase.Succinyl-CoA is then converted to succinic semialdehyde by aCoA-dependent aldehyde dehydrogenase. Alternately, succinate is directlyconverted to succinic semialdehyde by an acid reductase. The succinicsemialdehyde intermediate is reduced to 4-hydroxybutyrate (4-HB) by4-hydroxybutyrate dehydrogenase. A bifunctional aldehydedehydrogenase/alcohol dehydrogenase directly converts succinyl-CoA to4-HB. Activation of 4-HB to its acyl-CoA is catalyzed by a CoAtransferase or synthetase. Alternatively, 4-HB can be converted into a4-hydroxybutyryl-phosphate intermediate and subsequently transformedinto 4-HB-CoA by a phosphotrans-4-hydroxybutyrylase.

Isomerization of 4-hydroxybutyryl-CoA to 3-hydroxyisobutyryl-CoA iscatalyzed by a 4-HB-CoA methylmutase. Removal of the CoA moiety of3-hydroxyisobutyryl-CoA yields 3-hydroxyisobutyrate, which can besecreted as a product or further transformed to MAA by dehydration.Dehydration of 3-hydroxyisobutyryl-CoA to methacrylyl-CoA, followed byremoval of the CoA moiety by a CoA hydrolase, transferase or synthetase,is another route for MAA formation.

Exemplary enzyme candidates for the transformations shown in FIGS. 16Aand 16B are described below. Succinyl-CoA transferase and synthetaseenzymes were described previously in Example I (RTCA cycle).Methylmalonyl-CoA mutase and methylmalonyl-CoA epimerase were describedin Example III. 3-Hydroxyisobutyrate dehydratase enzyme candidates weredescribed in Example III.

Table 4 shows enzyme classes that can perform the steps depicted in FIG.16B. Exemplary enzymes are described in further detail below.

TABLE 4 Exemplary enzymes that carry out the steps in FIG. 16B. LabelFunction Step 1.1.1.a Oxidoreductase (oxo to alcohol) 5 1.1.1.c CoAreducatse (alcohol forming) 9 1.2.1.b CoA reductase (aldehyde forming) 41.2.1.e Oxidoreductase (acid to aldehyde) 8 2.3.1.a Phosphotransacylase7 2.7.2.a Kinase 6 2.8.3.a Coenzyme-A transferase   3, 10, 12, 153.1.2.a Thiolester hydrolase (CoA specific) 12, 15 4.2.1.a Dehydratase13, 14 5.4.99.a Methyl mutase 11  6.2.1.a Acid-thiol ligase/CoAsynthetase   3, 10, 12, 15

1.1.1.a.

Enzymes exhibiting 4-hydroxybutyrate dehydrogenase activity (EC1.1.1.61) reduce succinate semialdehyde to 4-hydroxybutyrate (Step 5 ofFIG. 16B). Such enzymes have been characterized in Ralstonia eutropha(Bravo et al., J Forens Sci, 49:379-387 (2004)), Clostridium kluyveri(Wolff et al., Protein Expr. Purif. 6:206-212 (1995)) and Arabidopsisthaliana (Breitkreuz et al., J Biol Chem, 278:41552-41556 (2003)). TheA. thaliana enzyme was cloned and characterized in yeast (Breitkreuz etal., J. Biol. Chem. 278:41552-41556 (2003)). Yet another gene is thealcohol dehydrogenase adhI from Geobacillus thermoglucosidasius (Jeon etal., J Biotechnol 135:127-133 (2008)).

PROTEIN GENBANK ID GI NUMBER ORGANISM 4hbd YP_726053.1 113867564Ralstonia eutropha H16 4hbd L21902.1 146348486 Clostridium kluyveri DSM555 4hbd Q94B07 75249805 Arabidopsis thaliana adhI AAR91477.1 40795502Geobacillus thermoglucosidasius

1.1.1.c.

A bifunctional enzyme with acyl-CoA reductase and alcohol dehydrogenaseactivity can directly convert succinyl-CoA to 4-hydroxybutyrate.Exemplary bifunctional oxidoreductases that convert an acyl-CoA toalcohol include those that transform substrates such as acetyl-CoA toethanol (for example, adhE from E. coli (Kessler et al., FEBS. Lett.281:59-63 (1991))) and butyryl-CoA to butanol (for example, adhE2 fromC. acetobutylicum (Fontaine et al., J. Bacteriol. 184:821-830 (2002))).The C. acetobutylicum enzymes encoded by bdh I and bdh II (Walter, etal., J. Bacteriol. 174:7149-7158 (1992)), reduce acetyl-CoA andbutyryl-CoA to ethanol and butanol, respectively. In addition toreducing acetyl-CoA to ethanol, the enzyme encoded by adhE inLeuconostoc mesenteroides has been shown to oxide the branched chaincompound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al., J. Gen.Appl. Microbiol. 18:43-55 (1972); Koo et al., Biotechnol Lett,27:505-510 (2005)). Another exemplary enzyme can convert malonyl-CoA to3-HP. An NADPH-dependent enzyme with this activity has characterized inChloroflexus aurantiacus where it participates in the3-hydroxypropionate cycle (Hugler et al., J Bacteriol, 184:2404-2410(2002); Strauss et al., Eur J Biochem, 215:633-643 (1993)). This enzyme,with a mass of 300 kDa, is highly substrate-specific and shows littlesequence similarity to other known oxidoreductases (Hugler et al.,supra). No enzymes in other organisms have been shown to catalyze thisspecific reaction; however there is bioinformatic evidence that otherorganisms may have similar pathways (Klatt et al., Env Microbiol,9:2067-2078 (2007)). Enzyme candidates in other organisms includingRoseiflexus castenholzii, Erythrobacter sp. NAP1 and marine gammaproteobacterium HTCC2080 can be inferred by sequence similarity.

Protein GenBank ID GI Number Organism adhE NP_415757.1 16129202Escherichia coli adhE2 AAK09379.1 12958626 Clostridium acetobutylicumadhE AAV66076.1 55818563 Leuconostoc mesenteroides bdh I NP_349892.115896543 Clostridium acetobutylicum bdh II NP_349891.1 15896542Clostridium acetobutylicum mcr AAS20429.1 42561982 Chloroflexusaurantiacus Rcas_2929 YP_001433009.1 156742880 Roseiflexus castenholziiNAP1_02720 ZP_01039179.1 85708113 Erythrobacter sp. NAP1 MGP2080_00535ZP_01626393.1 119504313 marine gamma proteobacterium HTCC2080

Longer chain acyl-CoA molecules can be reduced to their correspondingalcohols by enzymes such as the jojoba (Simmondsia chinensis) FAR whichencodes an alcohol-forming fatty acyl-CoA reductase. Its overexpressionin E. coli resulted in FAR activity and the accumulation of fattyalcohol (Metz et al., Plant Physiol, 122:635-644 (2000)).

Protein GenBank ID GI Number Organism FAR AAD38039.1 5020215 Simmondsiachinensis

1.2.1.b.

The reduction of succinyl-CoA to its corresponding aldehyde, succinatesemialdehyde, is catalyzed by succinyl-CoA reductase (aldehyde forming).Exemplary enzymes include succinyl-CoA reductase (EC 1.2.1.76),acetyl-CoA reductase, butyryl-CoA reductase and fatty acyl-CoAreductase. Enzymes with succinyl-CoA reductase activity are encoded bysucD of Clostridium kluyveri (Sohling, J. Bacteriol. 178:871-880 (1996))and sucD of P. gingivalis (Takahashi, J. Bacteriol 182:4704-4710(2000)). Additional succinyl-CoA reductase enzymes participate in the3-hydroxypropionate/4-hydroxybutyrate cycle of thermophilic archaeaincluding Metallosphaera sedula (Berg et al., Science 318:1782-1786(2007)) and Thermoproteus neutrophilus (Ramos-Vera et al., J Bacteriol,191:4286-4297 (2009)). The M. sedula enzyme, encoded by Msed_(—)0709, isstrictly NADPH-dependent and also has malonyl-CoA reductase activity.The T. neutrophilus enzyme is active with both NADPH and NADH. Theenzyme acylating acetaldehyde dehydrogenase in Pseudomonas sp, encodedby bphG, is yet another as it has been demonstrated to oxidize andacylate acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehydeand formaldehyde (Powlowski, J. Bacteriol. 175:377-385 (1993)). Inaddition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhEin Leuconostoc mesenteroides has been shown to oxidize the branchedchain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya, J. Gen.Appl. Microbiol. 18:43-55 (1972); and Koo et al., Biotechnol Lett.27:505-510 (2005)). Butyraldehyde dehydrogenase catalyzes a similarreaction, conversion of butyryl-CoA to butyraldehyde, in solventogenicorganisms such as Clostridium saccharoperbutylacetonicum (Kosaka et al.,Biosci Biotechnol Biochem., 71:58-68 (2007)). Exemplary fatty acyl-CoAreductases enzymes are encoded by acyl of Acinetobacter calcoaceticus(Reiser, Journal of Bacteriology 179:2969-2975 (1997)) and Acinetobactersp. M-1 (Ishige et al., Appl. Environ. Microbiol. 68:1192-1195 (2002)).

Protein GenBank ID GI Number Organism MSED_0709 YP_001190808.1 146303492Metallosphaera sedula Tneu_0421 Thermoproteus neutrophilus sucD P38947.1172046062 Clostridium kluyveri sucD NP_904963.1 34540484 Porphyromonasgingivalis bphG BAA03892.1 425213 Pseudomonas sp adhE AAV66076.155818563 Leuconostoc mesenteroides bld AAP42563.1 31075383 Clostridiumsaccharoperbutylacetonicum

Protein GenBank ID GI Number Organism acr1 YP_047869.1 50086359Acinetobacter calcoaceticus acr1 AAC45217 1684886 Acinetobacter baylyiacr1 BAB85476.1 18857901 Acinetobacter sp. Strain M-1

1.2.1.e.

Direct conversion of succinate to succinate semialdehyde is catalyzed bya carboxylic acid reductase. Exemplary enzymes include carboxylic acidreductase, alpha-aminoadipate reductase and retinoic acid reductase.Carboxylic acid reductase (CAR), found in Nocardia iowensis, catalyzesthe magnesium, ATP and NADPH-dependent reduction of carboxylic acids totheir corresponding aldehydes (Venkitasubramanian et al., J Biol. Chem.282:478-485 (2007)). The natural substrate of this enzyme is benzoateand the enzyme exhibits broad acceptance of aromatic substratesincluding p-toluate (Venkitasubramanian et al., Biocatalysis inPharmaceutical and Biotechnology Industries. CRC press (2006)). Theenzyme from Nocardia iowensis, encoded by car, was cloned andfunctionally expressed in E. coli (Venkitasubramanian et al., J Biol.Chem. 282:478-485 (2007)). CAR requires post-translational activation bya phosphopantetheine transferase (PPTase) that converts the inactiveapo-enzyme to the active holo-enzyme (Hansen et al., Appl. Environ.Microbiol 75:2765-2774 (2009)). Expression of the npt gene, encoding aspecific PPTase, product improved activity of the enzyme. An additionalenzyme candidate found in Streptomyces griseus is encoded by the griCand griD genes. This enzyme is believed to convert3-amino-4-hydroxybenzoic acid to 3-amino-4-hydroxybenzaldehyde asdeletion of either griC or griD led to accumulation of extracellular3-acetylamino-4-hydroxybenzoic acid, a shunt product of3-amino-4-hydroxybenzoic acid metabolism (Suzuki, et al., J. Antibiot.60(6):380-387 (2007)). Co-expression of griC and griD with SGR_(—)665,an enzyme similar in sequence to the Nocardia iowensis npt, can bebeneficial.

GenBank Gene Accession No. GI No. Organism car AAR91681.1 40796035Nocardia iowensis npt ABI83656.1 114848891 Nocardia iowensis griCYP_001825755.1 182438036 Streptomyces griseus griD YP_001825756.1182438037 Streptomyces griseus

Additional car and npt genes can be identified based on sequencehomology.

GenBank Gene name GI No. Accession No. Organism fadD9 121638475YP_978699.1 Mycobacterium bovis BCG BCG_2812c 121638674 YP_978898.1Mycobacterium bovis BCG nfa20150 54023983 YP_118225.1 Nocardia farcinicaIFM 10152 nfa40540 54026024 YP_120266.1 Nocardia farcinica IFM 10152SGR_6790 182440583 YP_001828302.1 Streptomyces griseus subsp. griseusNBRC 13350 SGR_665 182434458 YP_001822177.1 Streptomyces griseus subsp.griseus NBRC 13350 MSMEG_2956 YP_887275.1 YP_887275.1 Mycobacteriumsmegmatis MC2 155 MSMEG_5739 YP_889972.1 118469671 Mycobacteriumsmegmatis MC2 155 MSMEG_2648 YP_886985.1 118471293 Mycobacteriumsmegmatis MC2 155 MAP1040c NP_959974.1 41407138 Mycobacterium aviumsubsp. paratuberculosis K-10 MAP2899c NP_961833.1 41408997 Mycobacteriumavium subsp. paratuberculosis K-10 MMAR_2117 YP_001850422.1 183982131Mycobacterium marinum M MMAR_2936 YP_001851230.1 183982939 Mycobacteriummarinum M MMAR_1916 YP_001850220.1 183981929 Mycobacterium marinum MTpauDRAFT_33060 ZP_04027864.1 227980601 Tsukamurella paurometabola DSM20162 TpauDRAFT_20920 ZP_04026660.1 ZP_04026660.1 Tsukamurellapaurometabola DSM 20162 CPCC7001_1320 ZP_05045132.1 254431429 CyanobiumPCC7001 DDBDRAFT_0187729 XP_636931.1 66806417 Dictyostelium discoideumAX4

An enzyme with similar characteristics, alpha-aminoadipate reductase(AAR, EC 1.2.1.31), participates in lysine biosynthesis pathways in somefungal species. This enzyme naturally reduces alpha-aminoadipate toalpha-aminoadipate semialdehyde. The carboxyl group is first activatedthrough the ATP-dependent formation of an adenylate that is then reducedby NAD(P)H to yield the aldehyde and AMP. Like CAR, this enzyme utilizesmagnesium and requires activation by a PPTase. Enzyme candidates for AARand its corresponding PPTase are found in Saccharomyces cerevisiae(Morris et al., Gene 98:141-145 (1991)), Candida albicans (Guo et al.,Mol. Genet. Genomics 269:271-279 (2003)), and Schizosaccharomyces pombe(Ford et al., Curr. Genet. 28:131-137 (1995)). The AAR from S. pombeexhibited significant activity when expressed in E. coli (Guo et al.,Yeast 21:1279-1288 (2004)). The AAR from Penicillium chrysogenum acceptsS-carboxymethyl-L-cysteine as an alternate substrate, but did not reactwith adipate, L-glutamate or diaminopimelate (Hijarrubia et al., J Biol.Chem. 278:8250-8256 (2003)). The gene encoding the P. chrysogenum PPTasehas not been identified to date and no high-confidence hits wereidentified by sequence comparison homology searching.

GenBank Gene Accession No. GI No. Organism LYS2 AAA34747.1 171867Saccharomyces cerevisiae LYS5 P50113.1 1708896 Saccharomyces cerevisiaeLYS2 AAC02241.1 2853226 Candida albicans LYS5 AAO26020.1 28136195Candida albicans Lys1p P40976.3 13124791 Schizosaccharomyces pombe Lys7pQ10474.1 1723561 Schizosaccharomyces pombe Lys2 CAA74300.1 3282044Penicillium chrysogenum

2.3.1.a.

An enzyme with phosphotrans-4-hydroxybutyrylase activity is required toconvert 4-hydroxybutyryl-phosphate to 4-hydroxybutyryl-CoA. Exemplaryphosphate-transferring acyltransferases include phosphotransacetylase(EC 2.3.1.8) and phosphotransbutyrylase (EC 2.3.1.19). The pta gene fromE. coli encodes a phosphotransacetylase that reversibly convertsacetyl-CoA into acetyl-phosphate (Suzuki, Biochim. Biophys. Acta191:559-569 (1969)). This enzyme can also utilize propionyl-CoA as asubstrate, forming propionate in the process (Hesslinger et al., Mol.Microbiol 27:477-492 (1998)). Other phosphate acetyltransferases thatexhibit activity on propionyl-CoA are found in Bacillus subtilis (Radoet al., Biochim. Biophys. Acta 321:114-125 (1973)), Clostridium kluyveri(Stadtman, Methods Enzymol 1:596-599 (1955)), and Thermotoga maritima(Bock et al., J. Bacteriol. 181:1861-1867 (1999)). Similarly, the ptbgene from C. acetobutylicum encodes phosphotransbutyrylase, an enzymethat reversibly converts butyryl-CoA into butyryl-phosphate (Wiesenbornet al., Appl Environ. Microbiol 55:317-322 (1989); Walter et al., Gene134:107-111 (1993)). Additional ptb genes are found inbutyrate-producing bacterium L2-50 (Louis et al., J. Bacteriol.186:2099-2106 (2004)) and Bacillus megaterium (Vazquez et al., Curr.Microbiol 42:345-349 (2001)).

Protein GenBank ID GI Number Organism pta NP_416800.1 71152910Escherichia coli pta P39646 730415 Bacillus subtilis pta A5N801146346896 Clostridium kluyveri pta Q9X0L4 6685776 Thermotoga maritimaptb NP_349676 34540484 Clostridium acetobutylicum ptb AAR19757.138425288 butyrate-producing bacterium L2-50 ptb CAC07932.1 10046659Bacillus megaterium

2.7.2.a.

Kinase or phosphotransferase enzymes in the EC class 2.7.2 transformcarboxylic acids to phosphonic acids with concurrent hydrolysis of oneATP. Exemplary 4-Hydroxybutyrate kinase enzyme candidates includebutyrate kinase (EC 2.7.2.7), isobutyrate kinase (EC 2.7.2.14),aspartokinase (EC 2.7.2.4), acetate kinase (EC 2.7.2.1) andgamma-glutamyl kinase (EC 2.7.2.11). Butyrate kinase catalyzes thereversible conversion of butyryl-phosphate to butyrate duringacidogenesis in Clostridial species (Cary et al., Appl Environ Microbiol56:1576-1583 (1990)). The Clostridium acetobutylicum enzyme is encodedby either of the two buk gene products (Huang et al., J Mol. MicrobiolBiotechnol 2:33-38 (2000)). Other butyrate kinase enzymes are found inC. butyricum and C. tetanomorphum (Twarog et al., J. Bacteriol.86:112-117 (1963)). A related enzyme, isobutyrate kinase from Thermotogamaritima, was expressed in E. coli and crystallized (Diao et al., JBacteriol. 191:2521-2529 (2009); Diao et al., Acta Crystallogr. D. Biol.Crystallogr. 59:1100-1102 (2003)). Aspartokinase catalyzes theATP-dependent phosphorylation of aspartate and participates in thesynthesis of several amino acids. The aspartokinase III enzyme in E.coli, encoded by lysC, has a broad substrate range and the catalyticresidues involved in substrate specificity have been elucidated (Keng etal., Arch Biochem Biophys 335:73-81 (1996)). Two additional kinases inE. coli are also acetate kinase and gamma-glutamyl kinase. The E. coliacetate kinase, encoded by ackA (Skarstedt et al., J. Biol. Chem.251:6775-6783 (1976)), phosphorylates propionate in addition to acetate(Hesslinger et al., Mol. Microbiol 27:477-492 (1998)). The E. coligamma-glutamyl kinase, encoded by proB (Smith et al., J. Bacteriol.157:545-551 (1984)), phosphorylates the gamma carbonic acid group ofglutamate.

Protein GenBank ID GI Number Organism buk1 NP_349675 15896326Clostridium acetobutylicum buk2 Q97II1 20137415 Clostridiumacetobutylicum buk2 Q9X278.1 6685256 Thermotoga maritima lysCNP_418448.1 16131850 Escherichia coli ackA NP_416799.1 16130231Escherichia coli proB NP_414777.1 16128228 Escherichia coli

2.8.3.a.

CoA transferases catalyze the reversible transfer of a CoA moiety fromone molecule to another. Several transformations in FIG. 16B require aCoA transferase activity: succinyl-CoA transferase, 4-hydroxybutyryl-CoAtransferase, 3-hydroxyisobutyryl-CoA transferase and methacrylyl-CoAtransferase. Enzyme candidates for succinyl-CoA transferase have beendescribed previously herein and are also applicable to this pathway.Additional exemplary CoA transferase enzymes include the gene productsof cat1, cat2, and cat3 of Clostridium kluyveri which have been shown toexhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA transferaseactivity, respectively (Seedorf et al., Proc. Natl. Acad. Sci U.S.A105:2128-2133 (2008); Sohling et al., J Bacteriol. 178:871-880 (1996)).Similar CoA transferase activities are also present in Trichomonasvaginalis (van Grinsven et al., J. Biol. Chem. 283:1411-1418 (2008)) andTrypanosoma brucei (Riviere et al., J. Biol. Chem. 279:45337-45346(2004)).

Protein GenBank ID GI Number Organism cat1 P38946.1 729048 Clostridiumkluyveri cat2 P38942.2 172046066 Clostridium kluyveri cat3 EDK35586.1146349050 Clostridium kluyveri TVAG_395550 XP_001330176 123975034Trichomonas vaginalis G3 Tb11.02.0290 XP_828352 71754875 Trypanosomabrucei

The glutaconyl-CoA-transferase (EC 2.8.3.12) enzyme from anaerobicbacterium Acidaminococcus fermentans reacts with glutaconyl-CoA and3-butenoyl-CoA (Mack et al., 226:41-51 (1994)), substrates similar instructure to 2,3-dehydroadipyl-CoA. The genes encoding this enzyme aregctA and gctB. This enzyme has reduced but detectable activity withother CoA derivatives including glutaryl-CoA, 2-hydroxyglutaryl-CoA,adipyl-CoA, crotonyl-CoA and acrylyl-CoA (Buckel et al., Eur. J.Biochem. 118:315-321 (1981)). The enzyme has been cloned and expressedin E. coli (Mack et al., Eur. J. Biochem. 226:41-51 (1994)). GlutaconateCoA-transferase activity has also been detected in Clostridiumsporosphaeroides and Clostridium symbiosum. Additional glutaconateCoA-transferase enzymes can be inferred by homology to theAcidaminococcus fermentans protein sequence.

Protein GenBank ID GI Number Organism gctA CAA57199.1 559392Acidaminococcus fermentans gctB CAA57200.1 559393 Acidaminococcusfermentans gctA ACJ24333.1 212292816 Clostridium symbiosum gctBACJ24326.1 212292808 Clostridium symbiosum gctA NP_603109.1 19703547Fusobacterium nucleatum gctB NP_603110.1 19703548 Fusobacteriumnucleatum

A CoA transferase that can utilize acetyl-CoA as the CoA donor isacetoacetyl-CoA transferase, encoded by the E. coli atoA (alpha subunit)and atoD (beta subunit) genes (Korolev et al., Acta Crystallogr. D.Biol. Crystallogr. 58:2116-2121 (2002); Vanderwinkel et al., 33:902-908(1968)). This enzyme has a broad substrate range (Sramek et al., ArchBiochem Biophys 171:14-26 (1975)) and has been shown to transfer the CoAmoiety to acetate from a variety of branched and linear acyl-CoAsubstrates, including isobutyrate (Matthies et al., Appl Environ.Microbiol 58:1435-1439 (1992)), valerate (Vanderwinkel et al., Biochem.Biophys. Res. Commun. 33:902-908 (1968)) and butanoate (Vanderwinkel etal., Biochem. Biophys. Res. Commun. 33:902-908 (1968)). This enzyme isinduced at the transcriptional level by acetoacetate, so modification ofregulatory control may be necessary for engineering this enzyme into apathway (Pauli et al., Eur. J. Biochem. 29:553-562 (1972)). Similarenzymes exist in Corynebacterium glutamicum ATCC 13032 (Duncan et al.,68:5186-5190 (2002)), Clostridium acetobutylicum (Cary et al., ApplEnviron Microbiol 56:1576-1583 (1990); Wiesenborn et al., Appl EnvironMicrobiol 55:323-329 (1989)), and Clostridium saccharoperbutylacetonicum(Kosaka et al., Biosci. Biotechnol Biochem. 71:58-68 (2007)).

Gene GI # Accession No. Organism atoA 2492994 P76459.1 Escherichia coliatoD 2492990 P76458.1 Escherichia coli actA 62391407 YP_226809.1Corynebacterium glutamicum cg0592 62389399 YP_224801.1 Corynebacteriumglutamicum ctfA 15004866 NP_149326.1 Clostridium acetobutylicum ctfB15004867 NP_149327.1 Clostridium acetobutylicum ctfA 31075384 AAP42564.1Clostridium saccharoperbutylacetonicum ctfB 31075385 AAP42565.1Clostridium saccharoperbutylacetonicum

3.1.2.a.

3-Hydroxyisobutyryl-CoA hydrolase catalyzes the conversion of3-hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate. This enzymeparticipates in valine degradation pathways (Shimomura et al., J Biol.Chem. 269:14248-14253 (1994)). Genes encoding this enzyme include hibchof Rattus norvegicus (Shimomura et al., Methods Enzymol. 324:229-240(2000)) and Homo sapiens (Shimomura et al., supra). Similar genecandidates can also be identified by sequence homology, including hibchof Saccharomyces cerevisiae and BC_(—)2292 of Bacillus cereus.

GenBank Gene name Accession # GI# Organism hibch Q5XIE6.2 146324906Rattus norvegicus hibch Q6NVY1.2 146324905 Homo sapiens hibch P28817.22506374 Saccharomyces cerevisiae BC_2292 AP09256 29895975 Bacilluscereus

Methylmalonyl-CoA is converted to methylmalonate by methylmalonyl-CoAhydrolase (EC 3.1.2.7). This enzyme, isolated from Rattus norvegicusliver, is also active on malonyl-CoA and propionyl-CoA as alternativesubstrates (Kovachy et al., J. Biol. Chem., 258: 11415-11421 (1983)).The gene associated with this enzyme has not been identified to date.Exemplary CoA hydrolases with broad substrate ranges are suitablecandidates, as are the 3-HIB-CoA hydrolase enzymes described above. Theenzyme encoded by acot12 from Rattus norvegicus brain (Robinson et al.,Biochem. Biophys. Res. Commun. 71:959-965 (1976)) can react withbutyryl-CoA, hexanoyl-CoA and malonyl-CoA. The human dicarboxylic acidthioesterase, encoded by acot8, exhibits activity on glutaryl-CoA,adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl-CoA (Westin etal., J. Biol. Chem. 280:38125-38132 (2005)). The closest E. coli homologto this enzyme, tesB, can also hydrolyze a range of CoA thiolesters(Naggert et al., J Biol Chem 266:11044-11050 (1991)). A similar enzymehas also been characterized in the rat liver (Deana R., Biochem Int26:767-773 (1992)). Additional enzymes with hydrolase activity in E.coli include ybgC, paaI, and ybdB (Kuznetsova, et al., FEMS MicrobiolRev, 2005, 29(2):263-279; Song et al., J Biol Chem, 2006,281(16):11028-38). Though its sequence has not been reported, the enzymefrom the mitochondrion of the pea leaf has a broad substrate range, withdemonstrated activity on acetyl-CoA, propionyl-CoA, butyryl-CoA,palmitoyl-CoA, oleoyl-CoA, succinyl-CoA, and crotonyl-CoA (Zeiher etal., Plant. Physiol. 94:20-27 (1990)). The acetyl-CoA hydrolase, ACH1,from S. cerevisiae represents another candidate hydrolase (Buu et al.,J. Biol. Chem. 278:17203-17209 (2003)).

GenBank Gene name Accession # GI# Organism acot12 NP_570103.1 18543355Rattus norvegicus tesB NP_414986 16128437 Escherichia coli acot8CAA15502 3191970 Homo sapiens acot8 NP_570112 51036669 Rattus norvegicustesA NP_415027 16128478 Escherichia coli ybgC NP_415264 16128711Escherichia coli paaI NP_415914 16129357 Escherichia coli ybdB NP_41512916128580 Escherichia coli ACH1 NP_009538 6319456 Saccharomycescerevisiae

Yet another candidate hydrolase is the glutaconate CoA-transferase fromAcidaminococcus fermentans. This enzyme was transformed by site-directedmutagenesis into an acyl-CoA hydrolase with activity on glutaryl-CoA,acetyl-CoA and 3-butenoyl-CoA (Mack et al., FEBS. Lett. 405:209-212(1997)). This suggests that the enzymes encodingsuccinyl-CoA:3-ketoacid-CoA transferases and acetoacetyl-CoA:acetyl-CoAtransferases may also serve as candidates for this reaction step butwould require certain mutations to change their function.

GenBank Gene name Accession # GI# Organism gctA CAA57199 559392Acidaminococcus fermentans gctB CAA57200 559393 Acidaminococcusfermentans

4.2.1.a.

Two transformations in FIG. 16B require dehydratase enzymes. Thedehydration of 3-hydroxyisobutyrate to MAA is catalyzed by3-hydroxyisobutyrate. Enzyme candidates for this transformation aredescribed in Example VII. The dehydration of 3-hydroxyisobutyryl-CoA tomethacrylyl-CoA is catalyzed by an enzyme with 3-hydroxyisobutyryl-CoAdehydratase activity. Exemplary enzymes include enoyl-CoA hydratases andcrotonases.

Enoyl-CoA hydratases (EC 4.2.1.17) catalyze the dehydration of a rangeof 3-hydroxyacyl-CoA substrates (Roberts et al., Arch. Microbiol117:99-108 (1978); Agnihotri et al., Bioorg. Med. Chem. 11:9-20 (2003);Conrad et al., J. Bacteriol. 118:103-111 (1974)). The enoyl-CoAhydratase of Pseudomonas putida, encoded by ech, catalyzes theconversion of 3-hydroxybutyryl-CoA to crotonyl-CoA (Roberts et al.,Arch. Microbiol 117:99-108 (1978)). This transformation is alsocatalyzed by the crt gene product of Clostridium acetobutylicum, thecrt1 gene product of C. kluyveri, and other clostridial organisms Atsumiet al., Metab Eng 10:305-311 (2008); Boynton et al., J. Bacteriol.178:3015-3024 (1996); Hillmer et al., FEBS Lett. 21:351-354 (1972)).Additional enoyl-CoA hydratase candidates are phaA and phaB, of P.putida, and paaA and paaB from P. fluorescens (Olivera et al., Proc.Natl. Acad. Sci U.S.A 95:6419-6424 (1998)). The gene product of pimF inRhodopseudomonas palustris is predicted to encode an enoyl-CoA hydratasethat participates in pimeloyl-CoA degradation (Harrison et al.,Microbiology 151:727-736 (2005)). Lastly, a number of Escherichia coligenes have been shown to demonstrate enoyl-CoA hydratase functionalityincluding maoC (Park et al., J. Bacteriol. 185:5391-5397 (2003)), paaF(Ismail et al., Eur. J. Biochem. 270:3047-3054 (2003); Park et al.,Appl. Biochem. Biotechnol 113-116:335-346 (2004); Park et al.,Biotechnol Bioeng 86:681-686 (2004)) and paaG (Ismail et al., Eur. J.Biochem. 270:3047-3054 (2003); Park and Lee, Appl. Biochem. Biotechnol113-116:335-346 (2004); Park and Yup, Biotechnol Bioeng 86:681-686(2004)).

GenBank Gene Accession No. GI No. Organism ech NP_745498.1 26990073Pseudomonas putida crt NP_349318.1 15895969 Clostridium acetobutylicumcrt1 YP_001393856 153953091 Clostridium kluyveri phaA NP_745427.126990002 Pseudomonas putida phaB NP_745426.1 26990001 Pseudomonas putidapaaA ABF82233.1 106636093 Pseudomonas fluorescens paaB ABF82234.1106636094 Pseudomonas fluorescens maoC NP_415905.1 16129348 Escherichiacoli paaF NP_415911.1 16129354 Escherichia coli

GenBank Gene Accession No. GI No. Organism paaG NP_415912.1 16129355Escherichia coli

Alternatively, the E. coli gene products offadA and fadB encode amultienzyme complex involved in fatty acid oxidation that exhibitsenoyl-CoA hydratase activity (Yang et al., Biochemistry 30:6788-6795(1991); Yang, J Bacteriol. 173:7405-7406 (1991); Nakahigashi et al.,Nucleic Acids Res. 18:4937 (1990)). Knocking out a negative regulatorencoded by fadR can be utilized to activate the fadB gene product (Satoet al., J Biosci. Bioeng 103:38-44 (2007)). The fadI and fadJ genesencode similar functions and are naturally expressed under anaerobicconditions (Campbell et al., Mol. Microbiol 47:793-805 (2003)).

Protein GenBank ID GI Number Organism fadA YP_026272.1 49176430Escherichia coli fadB NP_418288.1 16131692 Escherichia coli fadINP_416844.1 16130275 Escherichia coli fadJ NP_416843.1 16130274Escherichia coli fadR NP_415705.1 16129150 Escherichia coli

5.4.99.a.

4-Hydroxybutyryl-CoA is rearranged to form 3-hydroxyisobutyryl-CoA by anenzyme with 4-hydroxybutyryl-CoA mutase activity. This activity has notbeen demonstrated to date. Methylmalonyl-CoA mutase and isobutyryl-CoAmutase catalyze similar transformations. Exemplary methylmalonyl-CoAmutase and isobutyryl-CoA mutase enzyme candidates are described inExamples V and VII.

6.2.1.a.

The conversion of acyl-CoA substrates to their acid products can becatalyzed by a CoA acid-thiol ligase or CoA synthetase in the 6.2.1family of enzymes. Enzymes of FIG. 16B in this class includesuccinyl-CoA synthetase, 4-hydroxybutyryl-CoA synthetase,3-hydroxyisobutyryl-CoA synthetase and methacrylyl-CoA synthetase.Succinyl-CoA synthetase enzyme candidates were described previously.Exemplary enzymes for catalyzing 4-hydroxybutyryl-CoA synthetase,3-hydroxyisobutyryl-CoA synthetase and methacrylyl-CoA synthetaseactivities are described herein and below. ADP-forming acetyl-CoAsynthetase (ACD, EC 6.2.1.13) is an enzyme that couples the conversionof acyl-CoA esters to their corresponding acids with the concomitantsynthesis of ATP. ACD I from Archaeoglobus fulgidus, encoded by AF1211,was shown to operate on a variety of linear and branched-chainsubstrates including isobutyrate, isopentanoate, and fumarate (Musfeldtet al., J. Bacteriol. 184:636-644 (2002)). A second reversible ACD inArchaeoglobus fulgidus, encoded by AF1983, was also shown to have abroad substrate range with high activity on cyclic compoundsphenylacetate and indoleacetate (Musfeldt and Schonheit, J. Bacteriol.184:636-644 (2002)). The enzyme from Haloarcula marismortui (annotatedas a succinyl-CoA synthetase) accepts propionate, butyrate, andbranched-chain acids (isovalerate and isobutyrate) as substrates, andwas shown to operate in the forward and reverse directions (Brasen etal., Arch Microbiol 182:277-287 (2004)). The ACD encoded by PAE3250 fromhyperthermophilic crenarchaeon Pyrobaculum aerophilum showed thebroadest substrate range of all characterized ACDs, reacting withacetyl-CoA, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA(Brasen et al, supra). Directed evolution or engineering can be used tomodify this enzyme to operate at the physiological temperature of thehost organism. The enzymes from A. fulgidus, H. marismortui and P.aerophilum have all been cloned, functionally expressed, andcharacterized in E. coli (Brasen and Schonheit, supra; Musfeldt andSchonheit, J. Bacteriol. 184:636-644 (2002)). An additional candidate issuccinyl-CoA synthetase, encoded by sucCD of E. coli and LSO and LSC2genes of Saccharomyces cerevisiae. These enzymes catalyze the formationof succinyl-CoA from succinate with the concomitant consumption of oneATP in a reaction which is reversible in vivo (Buck et al., Biochemistry24:6245-6252 (1985)). The acyl CoA ligase from Pseudomonas putida hasbeen demonstrated to work on several aliphatic substrates includingacetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoicacids and on aromatic compounds such as phenylacetic and phenoxyaceticacids (Fernandez-Valverde et al., Appl. Environ. Microbiol. 59:1149-1154(1993)). A related enzyme, malonyl CoA synthetase (6.3.4.9) fromRhizobium leguminosarum could convert several diacids, namely, ethyl-,propyl-, allyl-, isopropyl-, dimethyl-, cyclopropyl-,cyclopropylmethylene-, cyclobutyl-, and benzyl-malonate into theircorresponding monothioesters (Pohl et al., J. Am. Chem. Soc.123:5822-5823 (2001)).

Protein GenBank ID GI Number Organism AF1211 NP_070039.1 11498810Archaeoglobus fulgidus AF1983 NP_070807.1 11499565 Archaeoglobusfulgidus scs YP_135572.1 55377722 Haloarcula marismortui PAE3250NP_560604.1 18313937 Pyrobaculum aerophilum str. IM2 sucC NP_415256.116128703 Escherichia coli sucD AAC73823.1 1786949 Escherichia coli LSC1NP_014785 6324716 Saccharomyces cerevisiae LSC2 NP_011760 6321683Saccharomyces cerevisiae paaF AAC24333.2 22711873 Pseudomonas putidamatB AAC83455.1 3982573 Rhizobium leguminosarum

An active reductive TCA cycle improves the yield of the acetyl-CoAderived product methacrylic acid (MAA), as shown in the exemplarypathways of FIG. 18. Pathways of the reductive TCA cycle are describedherein as well as the conversion of succinate to 4-hydroxybutyryl-CoAand methacrylic acid (see also FIGS. 5 and 16B and Example VII). FIGS.18A and 18B show exemplary pathways. The enzymatic transformations arecarried out by the enzymes as shown. FIG. 18A shows the pathways forfixation of CO₂ to succinate using the reductive TCA cycle. FIG. 18Bshows exemplary pathways for the biosynthesis of 3-hydroxyisobutyricacid and methacrylic acid from succinate; the enzymatic transformationsshown are carried out by the following enzymes: A.3-Hydroxyisobutyryl-CoA dehydratase, B. Methacrylyl-CoA synthetase,transferase or hydrolase, C. Succinyl-CoA transferase or synthetase, D.Succinyl-CoA reductase (aldehyde forming), E. 4-Hydroxybutyratedehydrogenase, F. 4-Hydroxybutyrate kinase, G.Phosphotrans-4-hydroxybutyrylase, H. Succinate reductase, I.Succinyl-CoA reductase (alcohol forming), J. 4-Hydroxybutyryl-CoAsynthetase or transferase, K. 4-Hydroxybutyryl-CoA mutase, L.3-Hydroxyisobutyryl-CoA synthetase, transferase or hydrolase, M.3-Hydroxyisobutyrate dehydratase.

EXAMPLE XXI Pathways for the Production of MAA and 2-HydroxyisobutyricAcid from Acetyl-CoA

An active reductive TCA cycle improves the yield of the acetyl-CoAderived product methacrylic acid (MAA), as shown in the fluxdistribution of FIG. 16C. In this pathway, MAA is produced fromacetyl-CoA in five enzymatic steps. In the first step, two molecules ofacetyl-CoA are combined to form acetoacetyl-CoA. Acetoacetyl-CoA issubsequently reduced to 3-hydroxybutyryl-CoA. A methylmutase thenrearranges the carbon backbone of 3-hydroxybutyryl-CoA to2-hydroxyisobutyryl-CoA, which is then dehydrated to formmethacrylyl-CoA. Alternatively, 2-hydroxyisobutyryl-CoA can be convertedto 2-hydroxyisobutyrate, secreted, and recovered as product. The finalstep converting methacrylyl-CoA to MAA can be performed by a singleenzyme or a series of enzymes. An alternate strategy for convertingmethacrylyl-CoA into MAA entails a multi-step process in which MAA-CoAis converted to MAA via 3-hydroxyisobutyrate. By this process, MAA-CoAis first converted to 3-hydroxyisobutyryl-CoA, which can subsequently beconverted to 3-hydroxyisobutyrate by 3-hydroxyisobutyryl-CoAtransferase, synthetase or hydrolase. 3-Hydroxyisobutyrate can then beconverted to MAA biocatalytically via a 3-hydroxyisobutyratedehdyratase, or secreted and converted to MAA by chemical dehydration.

Enzyme candidates for each pathway step are described herein. CoAtransferase, synthetase and hydrolase enzymes catalyzing the conversionof methacrylyl-CoA to MAA were described above in Example VII. Enzymesdescribing the indirect conversion of methacrylyl-CoA to3-hydroxyisobutyrate and methacrylic acid are also described above.

Acetoacetyl-CoA Thiolase.

The formation of acetoacetyl-CoA from two acetyl-CoA units is catalyzedby acetyl-CoA thiolase. Exemplary enzymes are described in Example X.

EXAMPLE XXII Pathways for the Production of MAA from Pyruvate andAcetyl-CoA

An active reductive TCA cycle improves the yield of the acetyl-CoAderived product methacrylic acid (MAA). A flux distribution showing thisimproved yield is shown in FIG. 16D.

FIG. 16E (see also FIG. 1 and Example I) depicts exemplary pathways toMAA from acetyl-CoA and pyruvate via the intermediate citramalate. Alsoshown are pathways to MAA from aconitate. In one pathway acetyl-CoA andpyruvate are converted to citramalate by citramalate synthase.Dehydration of citramalate can yield either citraconate or mesaconate.Mesaconate and citraconate are interconverted by a cis/trans isomerase.Decarboxylation of mesaconate or citraconate yields MAA. In an alternatepathway, citramalate is formed from acetyl-CoA and pyruvate via acitramalyl-CoA intermediate, catalyzed by citramalyl-CoA lyase andcitramalyl-coA hydrolase, transferase or synthetase. Also shown arepathways from aconitate to MAA. In one pathway, aconitate is firstdecarboxylated to itaconate by aconitate decarboxylase. Itaconate isthen isomerized to citraconate by itaconate delta-isomerase. Conversionof citraconate to MAA proceeds either directly by decarboxylation orindirectly via mesaconate. In an alternate pathway, the itaconateintermediate is first converted to itaconyl-CoA by a CoA transferase orsynthetase. Hydration of itaconyl-CoA yields citramalyl-CoA, which canthen be converted to MAA as described previously.

Table 1 as provided in Example I shows enzyme classes that can performthe steps depicted in FIG. 16E. Exemplary enzymes are described belowand in further detail in Example I.

EC 2.3.1.a Synthase.

Citramalate synthase (EC 2.3.1.182) catalyzes the conversion ofacetyl-CoA and pyruvate to citramalate and coenzyme A. Exemplary enzymesare described in Example I.

EC 2.8.3.a CoA Transferase (Step E).

CoA transferases catalyze the reversible transfer of a CoA moiety fromone molecule to another. Two transformations in FIG. 16E utilize a CoAtransferase: conversion of citramalyl-CoA to citramalate and activationof itaconate to itaconyl-CoA. Citramalyl-CoA transferase (EC 2.8.3.7 and2.8.3.11) transfers a CoA moiety from citramalyl-CoA to a donor. Acitramalate:succinyl-CoA transferase enzyme is active in the3-hydroxypropionate cycle of glyoxylate assimilation. Exemplary enzymesare described in Example I.

EC 3.2.1.a CoA Hydrolase.

Enzymes in the 3.1.2 family hydrolyze acyl-CoA molecules to theircorresponding acids. Several CoA hydrolases with broad substrate rangesare suitable candidates for exhibiting citramalyl-CoA hydrolaseactivity. Exemplary enzymes are described in Example I.

EC 4.1.1.a Decarboxylase.

The final step of MAA synthesis in FIG. 16E is the decarboxylation ofeither mesaconate or citraconate. Exemplary enzymes are described inExample I.

EC 4.1.3.a Lyase.

Citramalyl-CoA lyase (EC 4.1.3.25) converts acetyl-CoA and pyruvate tocitramalyl-CoA. This enzyme participates in the 3-hydroxypropionate(3-HP) cycle of glyoxylate assimilation, where it acts in thecitramalyl-CoA degrading direction. Exemplary enzymes are described inExample I.

EC 4.2.1.a Dehydratase.

The dehydration of citramalate to citraconate is catalyzed by an enzymewith citramalate dehydratase (citraconate forming) activity (EC4.2.1.35). This enzyme, along with citramalate synthase, participates inthe threonine-independent isoleucine biosynthesis pathway characterizedin Methanocaldococcus jannaschii and Leptospira interrrogans. Thedehydration of citramalate in these organisms catalyzed byisopropylmalate isomerase (IPMI), which catalyzes both the dehydrationof citramalate to citraconate and the subsequent trans-addition of waterto citraconate to form methylmalate (Xu et al., J. Bacteriol.186:5400-5409 (2004); Drevland et al., J Bacteriol. 189:4391-4400(2007)). Exemplary enzymes are described in Example I.

EC 5.2.1.a Cis/Trans Isomerase.

The cis/trans isomerization of mesaconate and citraconate is catalyzedby an enzyme with citraconate isomerase activity. Suitable candidatesinclude aconitate isomerase (EC 5.3.3.7), maleate cis, trans-isomerase(EC 5.2.1.1), maleylacetone cis, trans-isomerase and cis,trans-isomerase of unsaturated fatty acids (Cti). Aconitate isomeraseinterconverts cis- and trans-aconitate. Exemplary enzymes are describedin Example I.

EC 5.3.3.a Delta-Isomerase.

The conversion of itaconate to citraconate is catalyzed by itaconatedelta-isomerase. Exemplary enzymes are described in Example I.

EC 6.2.1 CoA Synthetase.

The conversion of citramalyl-CoA to citramalate and itaconate toitaconyl-CoA can be catalyzed by a CoA acid-thiol ligase or CoAsynthetase in the 6.2.1 family of enzymes. Exemplary enzymes aredescribed in Example I.

An active reductive TCA cycle improves the yield of the acetyl-CoAderived product methacrylic acid (MAA), as shown in the exemplarypathways of FIG. 19. Pathways of the reductive TCA cycle are describedherein as well as the conversion of acetyl-CoA and/or pyruvate via theintermediate citramalate to methacrylic acid (see also FIGS. 1 and 16Eand Example I). FIGS. 19A and 19B show exemplary pathways. FIG. 19Ashows the pathways for fixation of CO₂ to acetyl-CoA and pyruvate usingthe reductive TCA cycle. FIG. 19B shows exemplary pathways for thebiosynthesis of methacrylate from acetyl-CoA and pyruvate; the enzymatictransformations shown are carried out by the following enzymes: 1.Citramalate synthase, 2. Citramalate dehydratase (citraconate forming),3. Citraconate decarboxylase, 4. Citramalyl-CoA lyase, 5. Citramalyl-CoAtransferase, synthetase or hydrolase, 6. Citramalate dehydratase(mesaconate forming), 7. Citraconate isomerase, 8. Mesaconatedecarboxylase, 9. Aconitate decarboxylase, 10. Itaconate isomerase, 11.Itaconyl-CoA transferase or synthetase, 12. Itaconyl-CoA hydratase.

An active reductive TCA cycle improves the yield of the acetyl-CoAderived product methacrylic acid (MAA), as shown in the exemplarypathways of FIG. 20. Pathways of the reductive TCA cycle are describedherein as well as the conversion of acetyl-CoA to methacrylic acid or2-hydroxyisobutyric acid (see also FIG. 8 and Example X). FIGS. 20A and20B show exemplary pathways. FIG. 20A shows the pathways for fixation ofCO₂ to acetyl-CoA using the reductive TCA cycle. FIG. 20B showsexemplary pathways for the biosynthesis of methacrylic acid and2-hydroxyisobutyric acid from acetyl-CoA.

An active reductive TCA cycle improves the yield of the acetyl-CoAderived product methacrylic acid (MAA), as shown in the exemplarypathways of FIG. 21. Pathways of the reductive TCA cycle are describedherein as well as the conversion of acetyl-CoA via the intermediate3-hydroxyisobutyryl-CoA to methacrylic acid (see also FIGS. 5, 8 and 9and Examples VII, X, XI and XIV). FIGS. 21A and 21B show exemplarypathways. FIG. 21A shows the pathways for fixation of CO₂ to acetyl-CoAusing the reductive TCA cycle. FIG. 21B shows exemplary pathways for thebiosynthesis of methacrylic acid and 3-hydroxyisobutyric acid fromacetyl-CoA; the enzymatic transformations shown are carried out by thefollowing enzymes: 1) Acetoacetyl-CoA thiolase (AtoB), 2)3-Hydroxybutyryl-CoA dehydrogenase (Hbd), 3) Crotonase (Crt), 4)Crotonyl-CoA hydratase (4-Budh), 5) 4-hydroxybutyryl-CoA mutase, 6)3-hydroxyisobutyryl-CoA hydrolase, synthetase, or transferase, 7)3-hydroxyisobutyric acid dehydratase, 8) 3-hydroxyisobutyryl-CoAdehydratase, 9) methacrylyl-CoA hydrolase, synthetase, or transferase.Crotonyl-CoA hydratase, forming 4-hydroxybutyrl-CoA, is the reversereaction of 4-hydroxybutyrl-CoA dehydratase, which catalyze thereversible conversion of 4-hydroxybutyryl-CoA to crotonyl-CoA (seeExample XIV). Thus, in an embodiment of the invention, a methacrylicacid pathway comprises acetoacetyl-CoA thiolase, acetoacetyl-CoAreductase, also referred to herein as 3-hydroxybutyryl-CoA dehydrogenase(see FIGS. 9 and 21), crotonase, 4-hydroxybutyryl-CoA dehydratase, alsoreferred to herein as crotonyl-CoA hydratase, 4-hydroxybutyryl-CoAmutase, 3-hydroxyisobutyryl-CoA synthetase or 3-hydroxyisobutyryl-CoAhydrolase or 3-hydroxyisobutyryl-CoA transferase, and3-hydroxyisobutyrate dehydratase. Alternatively, a methacrylic acidpathway comprises acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase,crotonase, 4-hydroxybutyryl-CoA dehydratase, 4-hydroxybutyryl-CoAmutase, 3-hydroxyisobutyryl-CoA dehydratase, and methacrylyl-CoAsynthetase or methacrylyl-CoA hydrolase or methacrylyl-CoA transferase(see FIG. 21).

EXAMPLE XXIII Exemplary Carboxylic Acid Reductases

This example describes the use of carboxylic acid reductases to carryout the conversion of a caroboxylic acid to an aldehyde.

1.2.1.e Acid Reductase.

The conversion of unactivated acids to aldehydes can be carried out byan acid reductase. Examples of such conversions include, but are notlimited, the conversion of 4-hydroxybutyrate, succinate,alpha-ketoglutarate, and 4-aminobutyrate to 4-hydroxybutanal, succinatesemialdehyde, 2,5-dioxopentanoate, and 4-aminobutanal, respectively. Onenotable carboxylic acid reductase can be found in Nocardia iowensiswhich catalyzes the magnesium, ATP and NADPH-dependent reduction ofcarboxylic acids to their corresponding aldehydes (Venkitasubramanian etal., J. Biol. Chem. 282:478-485 (2007)). This enzyme is encoded by thecar gene and was cloned and functionally expressed in E. coli(Venkitasubramanian et al., J. Biol. Chem. 282:478-485 (2007)).Expression of the npt gene product improved activity of the enzyme viapost-transcriptional modification. The npt gene encodes a specificphosphopantetheine transferase (PPTase) that converts the inactiveapo-enzyme to the active holo-enzyme. The natural substrate of thisenzyme is vanillic acid, and the enzyme exhibits broad acceptance ofaromatic and aliphatic substrates (Venkitasubramanian et al., inBiocatalysis in the Pharmaceutical and Biotechnology Industires, ed. R.N. Patel, Chapter 15, pp. 425-440, CRC Press LLC, Boca Raton, Fla.(2006)).

Gene Accession No. GI No. Organism car AAR91681.1 40796035 Nocardiaiowensis (sp. NRRL 5646) npt ABI83656.1 114848891 Nocardia iowensis (sp.NRRL 5646)

Additional car and npt genes can be identified based on sequencehomology.

Gene Accession No. GI No. Organism fadD9 YP_978699.1 121638475Mycobacterium bovis BCG BCG_2812c YP_978898.1 121638674 Mycobacteriumbovis BCG nfa20150 YP_118225.1 54023983 Nocardia farcinica IFM 10152nfa40540 YP_120266.1 54026024 Nocardia farcinica IFM 10152 SGR_6790YP_001828302.1 182440583 Streptomyces griseus subsp. griseus NBRC 13350SGR_665 YP_001822177.1 182434458 Streptomyces griseus subsp. griseusNBRC 13350

An additional enzyme candidate found in Streptomyces griseus is encodedby the griC and griD genes. This enzyme is believed to convert3-amino-4-hydroxybenzoic acid to 3-amino-4-hydroxybenzaldehyde asdeletion of either griC or griD led to accumulation of extracellular3-acetylamino-4-hydroxybenzoic acid, a shunt product of3-amino-4-hydroxybenzoic acid metabolism (Suzuki, et al., J. Antibiot.60(6):380-387 (2007)). Co-expression of griC and griD with SGR_(—)665,an enzyme similar in sequence to the Nocardia iowensis npt, can bebeneficial.

Gene Accession No. GI No. Organism griC 182438036 YP_001825755.1Streptomyces griseus subsp. griseus NBRC 13350 griD 182438037YP_001825756.1 Streptomyces griseus subsp. griseus NBRC 13350 MSMEG_2956YP_887275.1 YP_887275.1 Mycobacterium smegmatis MC2 155 MSMEG_5739YP_889972.1 118469671 Mycobacterium smegmatis MC2 155 MSMEG_2648YP_886985.1 118471293 Mycobacterium smegmatis MC2 155 MAP1040cNP_959974.1 41407138 Mycobacterium avium subsp. paratuberculosis K-10MAP2899c NP_961833.1 41408997 Mycobacterium avium subsp.paratuberculosis K-10 MMAR_2117 YP_001850422.1 183982131 Mycobacteriummarinum M MMAR_2936 YP_001851230.1 183982939 Mycobacterium marinum MMMAR_1916 YP_001850220.1 183981929 Mycobacterium marinum MTpauDRAFT_33060 ZP_04027864.1 227980601 Tsukamurella paurometabola DSM20162 TpauDRAFT_20920 ZP_04026660.1 227979396 Tsukamurella paurometabolaDSM 20162 CPCC7001_1320 ZP_05045132.1 254431429 Cyanobium PCC7001DDBDRAFT_0187729 XP_636931.1 66806417 Dictyostelium discoideum AX4

An enzyme with similar characteristics, alpha-aminoadipate reductase(AAR, EC 1.2.1.31), participates in lysine biosynthesis pathways in somefungal species. This enzyme naturally reduces alpha-aminoadipate toalpha-aminoadipate semialdehyde. The carboxyl group is first activatedthrough the ATP-dependent formation of an adenylate that is then reducedby NAD(P)H to yield the aldehyde and AMP. Like CAR, this enzyme utilizesmagnesium and requires activation by a PPTase. Enzyme candidates for AARand its corresponding PPTase are found in Saccharomyces cerevisiae(Morris et al., Gene 98:141-145 (1991)), Candida albicans (Guo et al.,Mol. Genet. Genomics 269:271-279 (2003)), and Schizosaccharomyces pombe(Ford et al., Curr. Genet. 28:131-137 (1995)). The AAR from S. pombeexhibited significant activity when expressed in E. coli (Guo et al.,Yeast 21:1279-1288 (2004)). The AAR from Penicillium chrysogenum acceptsS-carboxymethyl-L-cysteine as an alternate substrate, but did not reactwith adipate, L-glutamate or diaminopimelate (Hijarrubia et al., J.Biol. Chem. 278:8250-8256 (2003)). The gene encoding the P. chrysogenumPPTase has not been identified to date.

Gene Accession No. GI No. Organism LYS2 AAA34747.1 171867 Saccharomycescerevisiae LYS5 P50113.1 1708896 Saccharomyces cerevisiae LYS2AAC02241.1 2853226 Candida albicans LYS5 AAO26020.1 28136195 Candidaalbicans Lys1p P40976.3 13124791 Schizosaccharomyces pombe Lys7pQ10474.1 1723561 Schizosaccharomyces pombe Lys2 CAA74300.1 3282044Penicillium chrysogenum

Cloning and Expression of Carboxylic Acid Reductase.

Escherichia coli is used as a target organism to engineer the pathwayfor methacrylic acid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate. E. coli provides a good host for generating anon-naturally occurring microorganism capable of producing methacrylicacid, methacrylate ester, 3-hydroxyisobutyrate and/or2-hydroxyisobutyrate. E. coli is amenable to genetic manipulation and isknown to be capable of producing various intermediates and productseffectively under various oxygenation conditions.

To generate a microbial organism strain such as an E. coli strainengineered to produce methacrylic acid, methacrylate ester,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate], nucleic acidsencoding a carboxylic acid reductase and phosphopantetheine transferaseare expressed in E. coli using well known molecular biology techniques(see, for example, Sambrook, supra, 2001; Ausubel supra, 1999). Inparticular, car genes from Nocardia iowensis (designated 720),Mycobacterium smegmatis mc(2)155 (designated 890), Mycobacterium aviumsubspecies paratuberculosis K-10 (designated 891) and Mycobacteriummarinum M (designated 892) were cloned into pZS*13 vectors (Expressys,Ruelzheim, Germany) under control of PA1/lacO promoters. The npt(ABI83656.1) gene (i.e., 721) was cloned into the pKJL33S vector, aderivative of the original mini-F plasmid vector PML31 under control ofpromoters and ribosomal binding sites similar to those used in pZS*13.

The car gene (GNM_(—)720) was cloned by PCR from Nocardia genomic DNA.Its nucleic acid and protein sequences are shown in FIGS. 22A and 22B,respectively. A codon-optimized version of the npt gene (GNM_(—)721) wassynthesized by GeneArt (Regensburg, Germany). Its nucleic acid andprotein sequences are shown in FIGS. 23A and 23B, respectively. Thenucleic acid and protein sequences for the Mycobacterium smegmatismc(2)155 (designated 890), Mycobacterium avium subspeciesparatuberculosis K-10 (designated 891) and Mycobacterium marinum M(designated 892) genes and enzymes can be found in FIGS. 24, 25, and 26,respectively. The plasmids are transformed into a host cell to expressthe proteins and enzymes required for methacrylic acid, methacrylateester, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate production.

Additional CAR variants were generated. A codon optimized version of CAR891 was generated and designated 891 GA. The nucleic acid and amino acidsequences of CAR 891GA are shown in FIGS. 27A and 27B, respectively.Over 2000 CAR variants were generated. In particular, all 20 amino acidcombinations were made at positions V295, M296, G297, G391, G421, D413,G414, Y415, G416, and S417, and additional variants were tested as well.Exemplary CAR variants include: E16K; Q95L; L100M; A1011T; K823E; T941S;H15Q; D198E; G446C; S392N; F699L; V883I; F467S; T987S; R12H; V295G;V295A; V295S; V295T; V295G; V295V; V295L; V295I; V295M; V295P; V295F;V295Y; V295W; V295D; V295E; V295N; V295Q; V295H; V295K; V295R; M296G;M296A; M296S; M296T; M296C; M296V; M296L; M296I; M296M; M296P; M296F;M296Y; M296W; M296D; M296E; M296N; M296Q; M296H; M296K; M296R; G297G;G297A; G297S; G297T; G297C; G297V; G297L; G297I; G297M; G297P; G297F;G297Y; G297W; G297D; G297E; G297N; G297Q; G297H; G297K; G297R; G391G;G391A; G391S; G391T; G391C; G391V; G391L; G391I; G391M; G391P; G391F;G391Y; G391W; G391D; G391E; G391N; G391Q; G391H; G391K; G391R; G421G;G421A; G421S; G421T; G421C; G421V; G421L; G421I G421M; G421P; G421F;G421Y; G421W; G421D; G421E; G421N; G421Q; G421H; G421K; G421R; D413G;D413A; D413S; D413T; D413C; D413V; D413L; D413I; D413M; D413P; D413F;D413Y; D413W; D413D; D413E; D413N; D413Q; D413H; D413K; D413R; G414G;G414A; G414S; G414T; G414C; G414V; G414L; G414I; G414M; G414P; G414F;G414Y; G414W; G414D; G414E; G414N; G414Q; G414H; G414K; G414R; Y415G;Y415A; Y415S; Y415T; Y415C; Y415V; Y415L; Y415I; Y415M; Y415P; Y415F;Y415Y; Y415W; Y415D; Y415E; Y415N; Y415Q; Y415H; Y415K; Y415R; G416G;G416A; G416S; G416T; G416C; G416V; G416L; G416I; G416M; G416P; G416F;G416Y; G416W; G416D; G416E; G416N; G416Q; G416H; G416K; G416R; S417G;S417A; S417S; S417T; S417C; S417V S417L; S417I; S417M; S417P; S417F;S417Y; S417W; S417D; S417E; S417N; S417Q; S417H; S417K; and S417R.

The CAR variants were screened for activity, and numerous CAR variantswere found to exhibit CAR activity.

This example describes the use of CAR for converting carboxylic acids toaldehydes.

EXAMPLE XXIV Pathway for the Conversion of 4-Hydroxybutyryl-CoA toMethyl Methacrylate

This example describes a pathway for converting 4-hydroxybutyryl-CoA tomethyl methacrylate as an exemplary methacrylate ester.

An exemplary pathway to methyl methacrylate is shown in FIG. 30.Briefly, 4-hydroxybutyryl-CoA can be converted to 3-hydroxybutyryl-CoAas described above. 3-Hydroxyisobutyryl-CoA can be converted to3-hydroxyisobutyrate methyl ester as shown in FIG. 30 using an alcoholtranferase, as described above, in particular a methyl transferase (seeExample III; see also WO/2007/039415 and U.S. Pat. No. 7,901,915).3-Hydroxyisobutyrate methyl ester can be converted to methylmethacrylate by a dehydratase or by chemical conversion, as describedabove, for example, for the conversion of methacrylate to methacrylateesters (see FIG. 2). Exemplary dehydratases include those describedabove, for example, in Examples V, VI, X and XII.

Nucleic acids (especially a DNA) coding for an enzyme capable ofeffecting the transfer of an alcohol moietyl from an alcohol startingmaterial as defined herein to (meth)acrylyl CoA under removal of the CoAmoiety, such as a transferase (EC 2) or hydrolase (EC 3) class ofenzymes, e.g. lipase, esterase (such as acetyl choline esterase),transferase (such as choline acetyl transferase), protease or acylase(such as aminoacylase) (including coding for one or more such enzymes).

An EC group 2 enzyme, that is a transferase includes, for example,transaminases, transamidases, transketolases, transphosphorylases orcholine acetyl transferase that are known to catalyse the movement of achemical group from one compound to another. An EC class 3; hydrolase,which are also capable of catalysing transesterification reactions,include, for example, lipases from Candida antartica (CAL B), porcinepancreatic lipase (PPL), Candid rugosa (CRL) Pseudomonas cepacia (PCL)and the like; esterases such as pig liver esterase (PLE) acetyl cholineesterase; also proteases such as subtilisin, protease, for instance fromAspergillus spp., Bacillus spp., Rhizopus spp and other hydrolases suchas penicillin amidase or, for example, L-amino acylase can be suitableenzymes to catalyse the transfer.

These enzymes can be derived from a prokaryote such as a bacterium, aeukaryote or a higher organism, such as lipase from Candida spp.,esterase or choline acetyl transferase from mammalian cells, and theycan be supplied to the reaction as such. Alternatively, it can also beproduced biosynthetically from appropriate starting materials derivedfrom biomass.

While this example describes conversion to methyl methacrylate, it isunderstood by those skilled in the art that other methacrylate esterscan be made using a similar pathway and selecting an appropriatetransferase for the desired ester.

EXAMPLE XXV Conversion of 3-Hydroxyisobutyrate, 2-Hydroxyisobutyrate,3-Hydroxyisobutyryl-CoA or 2-Hyrdoxyisobutyryl-CoA to a MethacrylateEster

This example describes the conversion of 3-hydroxyisobutyrate,2-hydroxyisobutyrate, 3-hydroxyisobutyryl-CoA or 2-hydroxyisobutyryl-CoAto a methacrylate ester.

Alternative scenarios included herein are:

-   -   (1) A. Sugars to 3-hydroxyisobutyryl-CoA (enzymatic, pathways        described previously), B. 3-hydroxyisobutyryl-CoA to        3-hydroxyisobutyrate ester by an alcohol transferase, C.        3-hydroxyisobutyrate ester to methacrylate ester by a        dehydratase enzyme or chemical conversion    -   (2) A. Sugars to 2-hydroxyisobutyryl-CoA (enzymatic, pathways        described previously), B. 2-hydroxyisobutyryl-CoA to        2-hydroxyisobutyrate ester by an alcohol transferase, C.        2-hydroxyisobutyrate ester to methacrylate ester by a        dehydratase enzyme or chemical conversion    -   (3) A. Sugars to 3-hydroxyisobutyrate (enzymatic, pathway        described previously), B. 3-hydroxyisobutyrate to        3-hydroxyisobutyrate ester by a 3-hydroxyisobutyrate ester        forming enzyme, C. 3-hydroxyisobutyrate ester to methacrylate        ester by a dehydratase enzyme or chemical conversion    -   (4) A. Sugars to 2-hydroxyisobutyrate (enzymatic, pathway        described previously), B. 2-hydroxyisobutyrate to        2-hydroxyisobutyrate ester by a 2-hydroxyisobutyrate ester        forming enzyme, C. 2-hydroxyisobutyrate ester to methacrylate        ester by a dehydratase enzyme or chemical conversion    -   (5) A. Exogenous 3-hydroxyisobutyrate supplied to organism        (could be a product of another organism (i.e. co-culture or        separate fermentation) or could be derived from other        sources), B. 3-hydroxyisobutyrate to 2-hydroxyisobutyrate ester        by a 3-hydroxyisobutyrate ester forming enzyme, C.        3-hydroxyisobutyrate ester to methacrylate ester by a        dehydratase enzyme or chemical conversion    -   (6) A. Exogenous 3-hydroxyisobutyrate supplied to organism        (could be a product of another organism (i.e. co-culture or        separate fermentation) or could be derived from other        sources), B. 3-hydroxyisobutyrate to 3-hydroxyisobutyryl-CoA by        a CoA transferase or synthetase, C. formation of        3-hydroxyisobutyrate ester by an alcohol transferase enzyme, C.        3-hydroxyisobutyrate ester to methacrylate ester by a        dehydratase enzyme or chemical conversion    -   (7) A. Exogenous 2-hydroxyisobutyrate supplied to organism        (could be a product of another organism (i.e. co-culture or        separate fermentation) or could be derived from other        sources), B. 2-hydroxyisobutyrate to 2-hydroxyisobutyrate ester        by a 2-hydroxyisobutyrate ester forming enzyme, C.        2-hydroxyisobutyrate ester to methacrylate ester by a        dehydratase enzyme or chemical conversion    -   (8) A. Exogenous 2-hydroxyisobutyrate supplied to organism        (could be a product of another organism (i.e. co-culture or        separate fermentation) or could be derived from other        sources), B. 2-hydroxyisobutyrate to 2-hydroxyisobutyryl-CoA by        a CoA transferase or synthetase, C. formation of        2-hydroxyisobutyrate ester by an alcohol transferase enzyme, C.        2-hydroxyisobutyrate ester to methacrylate ester by a        dehydratase enzyme or chemical conversion

Pathways to methacrylate esters from 3-hydroxybutyrate,2-hydroxybutyrate, 3-hydroxyisobutyryl-CoA and 2-hydroxyisobutyryl-CoAare shown in FIGS. 28 and 29. Examplary pathways to these methacrylateester precursors have been described previously in this application andare shown in FIGS. 3, 5 and 8. 3-Hydroxybutyrate can be formed fromsuccinyl-CoA by the pathway shown in FIG. 3 or from 4-hydroxybutyryl-CoAas shown in FIG. 5. 3-Hydroxybutyryl-CoA can be formed from4-hydroxybutyryl-CoA by the pathway shown in FIG. 5.2-Hydroxyisobutyrate and 2-hydroxybutyryl-CoA can be formed by thepathway described in FIG. 8.

2-Hydroxyisobutyrate and its CoA ester, 2-hydroxyisobutyryl-CoA, can beinterconverted by a 2-hydroxyisobutyryl-CoA transferase or2-hydroxyisobutyryl-CoA synthetase. Likewise, 3-hydroxyisobutyrate andits CoA ester, 3-hydroxyisobutyryl-CoA, can be interconverted by a3-hydroxyisobutyryl-CoA transferase or 3-hydroxyisobutyryl-CoAsynthetase. Exemplary CoA transferase and synethetase enzymes aredescribed above in Examples I and VII.

The dehydration of 3-hydroxyisobutyric ester to methacrylic ester can beperformed under similar conditions as the dehydration of3-hyroxyisobutyric acid to methacrylic acid. These reaction conditionsare mild (reference: US Patent application 20100068773).

The chemical dehydration of 2-hydroxyisobutyrate ester to methacrylateester is analogous to the dehydration of 2-hydroxyisobutyric acid tomethacrylic acid, which is disclosed, for example, in U.S. Pat. Nos.3,666,805 and 5,225,594. In these references, 2-hydroxyisobutryic acidis dehydrated using metal oxides and hydroxides, ion exchange resins,alumina, silica, amines, phosphines, alkali metal alkoxides orcarboxylates, at reaction temperatures typically between 160-250 degreesC. In one method (U.S. Pat. No. 5,225,594), 2-hydroxyisobutyric acid andsodiumhydroxide were reacted at 185-195 degrees C. under vacuum (300torr) with stirring, resulting in a 97.1% conversion of2-hydroxyisobutyric acid, and a 96% yield of methacrylic acid. A similarapproach could be applied to dehydrate 2-hydroxyisobutyric ester tomethacrylic ester.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties, includingGenBank and GI number publications, are hereby incorporated by referencein this application in order to more fully describe the state of the artto which this invention pertains. Although the invention has beendescribed with reference to the examples provided above, it should beunderstood that various modifications can be made without departing fromthe spirit of the invention.

What is claimed is:
 1. A non-naturally occurring bacterial organismhaving a methacrylic acid pathway, said bacterial organism comprising atleast one exogenous nucleic acid encoding a methacrylic acid pathwayenzyme expressed in a sufficient amount to produce methacrylic acid,said methacrylic acid pathway comprising a pathway selected from: (a)citramalate synthase, citraconate-forming citramalate dehydratase, and adecarboxylase of a C1-C6 carboxylate; (b) citramalate synthase,citraconate-forming citramalate dehydratase, citraconate isomerase, anda decarboxylase of a C1-C6 carboxylate; (c) citramalate synthase,mesaconate-forming citramalate dehydratase, citraconate isomerase, and adecarboxylase of a C1-C6 carboxylate; (d) citramalate synthase,mesaconate-forming citramalate dehydratase, and a decarboxylase of aC1-C6 carboxylate; (e) citramalyl-CoA lyase, citramalyl-CoA transferase,synthetase or hydrolase, citraconate-forming citramalate dehydratase anda decarboxylase of a C1-C6 carboxylate; (f) citramalyl-CoA lyase,citramalyl-CoA transferase, synthetase or hydrolase, citraconate-formingcitramalate dehydratase citraconate isomerase, and a decarboxylase of aC1-C6 carboxylate; (g) citramalyl-CoA lyase, citramalyl-CoA transferase,synthetase or hydrolase, mesaconate-forming citramalate dehydratase, anda decarboxylase of a C1-C6 carboxylate; (h) citramalyl-CoA lyase,citramalyl-CoA transferase, synthetase or hydrolase, mesaconate-formingcitramalate dehydratase, citraconate isomerase, and a decarboxylase of aC1-C6 carboxylate; (i) aconitate decarboxylase, itaconate isomerase, anda decarboxylase of a C1-C6 carboxylate; (j) aconitate decarboxylase,itaconate isomerase, citraconate isomerase, and a decarboxylase of aC1-C6 carboxylate; (k) aconitate decarboxylase, itaconyl-CoAtransferase, synthetase or hydrolase, citramalyl-CoA dehydratase,citramalyl-CoA transferase, synthetase or hydrolase, citraconate-formingcitramalate dehydratase, and a decarboxylase of a C1-C6 carboxylate; (l)aconitate decarboxylase, itaconyl-CoA transferase, synthetase orhydrolase, citramalyl-CoA dehydratase, citramalyl-CoA transferase,synthetase or hydrolase, citraconate-forming citramalate dehydratase,citraconate isomerase, and a decarboxylase of a C1-C6 carboxylate; (m)aconitate decarboxylase, itaconyl-CoA transferase, synthetase orhydrolase, citramalyl-CoA dehydratase, citramalyl-CoA transferase,synthetase or hydrolase, mesaconate-forming citramalate dehydratase, anda decarboxylase of a C1-C6 carboxylate; and (n) aconitate decarboxylase,itaconyl-CoA transferase, synthetase or hydrolase, citramalyl-CoAdehydratase, citramalyl-CoA transferase, synthetase or hydrolase,mesaconate-forming citramalate dehydratase, citraconate isomerase, and adecarboxylase of a C1-C6 carboxylate.
 2. The non-naturally occurringbacterial organism of claim 1, wherein said non-naturally occurringbacterial organism further comprises a methacrylate ester pathwaycomprising at least one exogenous nucleic acid encoding a methacrylateester pathway enzyme expressed in a sufficient amount to produce amethacrylate ester, said methacrylate ester pathway comprisingmethacrylyl-CoA synthetase, methacrylyl-CoA transferase, and alcoholtransferase.
 3. A method for producing methacrylic acid, comprisingculturing the non-naturally occurring bacterial organism of claim 1under conditions and for a sufficient period of time to producemethacrylic acid.
 4. The non-naturally occurring bacterial organism ofclaim 1 wherein the decarboxylase is a decarboxylase of a C1-C6carboxylate comprising unsaturation.
 5. The non-naturally occurringbacterial organism of claim 1 wherein the decarboxylase is adecarboxylase of a C1-C6 carboxylate having two carboxylate groups. 6.The non-naturally occurring bacterial organism of claim 1 wherein thedecarboxylase is selected from the group consisting of aconitatedecarboxylase, 4-oxalocrotonate decarboxylase, cinnamate decarboxylase,4-oxalocronate decarboxylase, and sorbic acid decarboxylase.
 7. Thenon-naturally occurring bacterial organism of claim 1 which isEscherichia coli.